Human cord blood as a source of neural tissue for repair of the brain and spinal cord

ABSTRACT

The present invention relates to the use of umbilical cord blood cells from a donor or patient to provide neural cells which may be used in transplantation. The isolated cells according to the present invention may be used to effect autologous and allogeneic transplantation and repair of neural tissue, in particular, tissue of the brain and spinal cord and to treat neurodegenerative diseases of the brain and spinal cord.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of human umbilical cordblood and/or mononuclear cell fragment, thereof from a donor or patientto provide neural cells for use in transplantation. The isolated cellsaccording to the present invention may be used to effect transplantationand repair of neural tissue, in particular, tissue of the brain andspinal cord and to treat neurodegenerative diseases and injury of thebrain and spinal cord.

BACKGROUND OF THE INVENTION

[0002] Neurobiologists believe that the neurons in the adult brain andspinal cord are impossible to rebuild once they are damaged. Thus,science provided little hope to patients suffering from brain and spinalcord injury or from neurodegenerative diseases such as Alzheimer'sdisease and Parkinson's disease, among a number of others. Parkinson'sand Alzheimer's diseases are examples of neurodegenerative diseaseswhich are thought to be untreatable.

[0003] Parkinson's disease (PD), is a disorder of middle or late life,with very gradual progression and a prolonged course. HARRISON'SPRINCIPLES OF INTERNAL MEDICINE, Vol. 2, 23d ed., Ed by Isselbacher,Braunwald, Wilson, Martin, Fauci and Kasper, McGraw-Hill Inc., New YorkCity, 1994, pg. 2275. The most regularly observed changes in patientswith Parkinson's disease have been in the aggregates ofmelanin-containing nerve cells in the brainstem (substantia nigra, locus20 coeruleus), where there are varying degrees of nerve cell loss withreactive gliosis (most pronounced in the substantia nigra) along withdistinctive eosinophilic intracytoplasmic inclusions. (Id. at 2276). Inits fully developed form, PD is easily recognized in patients, wherestooped posture, stiffness and slowness of movement, fixity of facialexpression, rhythmic tremor of the limbs, which subsides on activewilled movement or complete relaxation, are common features. Generally,accompanying the other characteristics of the fully developed disorderis the festinating gait, whereby the patient, progresses or walks withquick shuffling steps at an accelerating pace as if to catch up with thebody's center of gravity. (ld. at 2276).

[0004] The treatment of Parkinson's disease pharmacologically withlevodopa combined with stereotactic surgery has only represented apartial cure, at best. (Id. at 2277). Underlying much of the treatmentdifficulty is directed to the fact that none of these therapeuticmeasures has an effect on the underlying disease process, which consistsof neuronal degeneration. Ultimately, a point seems to be reached wherepharmacology can no longer compensate for the loss of basal gangliadopamine. (Id.).

[0005] Alzheimer's Disease (AD) is caused by a degenerative process inthe patient which is characterized by progressive loss of cells from thebasal forebrain, cerebral cortex and other brain areas. Acetylcholinetransmitting neurons and their target nerves are particularly affected.Senile plaques and neurofibrillary tangles are present. Pick's diseasehas a similar clinical picture to Alzheimer's disease but a somewhatslower clinical course and circumscribed atrophy, mainly affecting thefrontal and temporal lobes. One animal model for Alzheimer's disease andother dementias displays hereditary tendency toward the formation ofsuch plaques. It is thought that if a drug has an effect in the model,it also may be beneficial in at least some forms of Alzheimer's andPick's diseases. At present there are palliative treatments but no meansto restore function in Alzheimer's patients.

[0006] A group of related neuronal degenerative disorders ischaracterized by progressive ataxia due to degeneration of thecerebellum, brainstem, spinal cord and peripheral nerves, andoccasionally the basal ganglia. Many of these syndromes are hereditary;others occur sporadically. The spinocerebellar degenerations arelogically placed in three groups: predominantly spinal ataxias,cerebellar ataxias and multiple-system degenerations. To date there areno treatments. Friedrich's ataxia is the prototypical spinal ataxiawhose inheritance is autosomal recessive. The responsible gene has beenfound on Chromosome 9. Symptoms begin between ages of 5 and 15 withunsteady gait, followed by upper extremity ataxia and dysarthria.Patients are flexic and lose large-fiber sensory modalities (vibrationand position sense). Two other diseases have similar symptoms:Bassen-Kornzweig syndrome (abeta-lipoproteinemia and vitamin Edeficiency) and Refsom's disease (phytanic acid storage disease).Cerebellar cortical degenerations generally occur between ages 30 and50. Clinically only signs of cerebellar dysfunction can be detected,with pathologic changes restricted to the cerebellum and occasionallythe inferior olives. Inherited and sporadic cases have been reported.Similar degeneration may also be associated with chronic alcoholism. Inmultiple-system degenerations, ataxia occurs in young to middle adultlife in varying combinations with spasticity and extrapyramidal,sensory, lower motor neuron and autonomic dysfunction. In some families,there may also be optic atrophy, retinitis pigmentosa, ophthalmoplegiaand dementia.

[0007] Another form of cerebellar degeneration is paraneoplasticcerebellar degeneration that occurs with certain cancers, such as oatcell lung cancer, breast cancer and ovarian cancer. In some cases, theataxia may precede the discovery of the cancer by weeks to years.Purkinje cells are permanently lost, resulting in ataxia. Even if thepatient is permanently cured of the cancer, their ability to functionmay be profoundly disabled by the loss of Purkinje cells. There is nospecific treatment.

[0008] Strokes also result in neuronal degeneration and loss offunctional synapses. Currently there is no repair, and only palliationand rehabilitation are undertaken.

[0009] Neurotransplantation has been used to explore the development ofthe central nervous system and for repair of diseased tissue inconditions such as Parkinson's and other neurodegenerative diseases. Theexperimental replacement of neurons by direct grafting of fetal tissueinto the brain has been accomplished in small numbers of patients inseveral research universities (including the University of SouthFlorida); but so far, the experimental grafting of human fetal neuronshas been limited by scarcity of appropriate tissue sources, logisticproblems, legal and ethical constraints, and poor survival of graftedneurons in the human host brain. One method replaces neurons by usingbone marrow stromal cells as stem cells for non-hematopoietic tissues.Marrow stromal cells can be isolated from other cells in marrow by theirtendency to adhere to tissue culture plastic. The cells have many of thecharacteristics of stem cells for tissues that can roughly be defined:as mesenchyrnal, because they can be differentiated in culture intoosteoblasts, chondrocytes, adipocytes, and even myoblasts. Thispopulation of bone marrow cells (BMSC) have also been used to preparedendritic cells, (K. Inaba, et al., J Experimental Med. 176: 1693-1702(1992)) which, as the name implies, have a morphology which might beconfused for neurons. Dendritic cells comprise a system ofantigen-presenting cells involved in the initiation of T cell responses.The specific growth factor, which stimulates production of dendriticcells, has been reported to be granulocyte/macrophage colony-stimulatingfactor 30 (GM-CSF). K. Inaba, et al., J Experimental Med. 176: 1693-1702(1992).

[0010] Work has recently been performed using stem cells obtained frombone marrow to provide neural cells which can be used in neuronaltransplantation. See WO 99/56759. This patent represented theculmination of more than 130 years of work in the use of bone marrowstem cells for non-hematopoietic uses.

[0011] Several groups of investigators since 1990 have attempted toprepare more homogenous populations of stem cells from bone marrow. Forexample, U.S. Pat. No. 5,087,570, issued Feb. 11, 1992, discloses how toisolate homogeneous mammalian hematopoietic stem cell compositions.Concentrated hematopoietic stem cell compositions are substantially freeof differentiated or dedicated hematopoietic cells. The desired cellsare obtained by subtraction of other cells having particular markers.The resulting composition may be used to provide for individual orgroups of hematopoietic lineages, to reconstitute stem cells of thehost, and to identify an assay for a wide variety of hernatopoieticgrowth factors.

[0012] U.S. Pat. No. 5,633.426 issued May 27, 1997, is another exampleof the differentiation and production of hematopoietic cells. Chimericimmunocompromised mice are given human bone marrow of at least 4 weeksfrom the time of implantation. The bone marrow assumed the normalpopulation of bone marrow except for erythrocytes. These mice with humanbone marrow may be used to study the effect of various agents on theproliferation and differentiation of human hematopoietic cells.

[0013] U.S. Pat. No. 5,665,557, issued Sep. 9, 1997, relates to methodsof obtaining concentrated hematopoietic stem cells by separating out anenriched fraction of cells expressing the marker CDw 109. Methods ofobtaining compositions enriched in hematopoietic megakaryocyteprogenitor cells are also provided. Compositions enriched for stem cellsand populations of cells obtained therefrom are also provided by theinvention. Methods of use of the cells are also included.

[0014] U.S. Pat. No. 5,453,505 issued on Jun. 5, 1995, is yet anothermethod of differentiation. Primordial tissue is introduced intoimmunodeficient hosts, where the primordial tissue develops anddifferentiates. The chimeric host allows for investigation of theprocesses and development of the xenogeneic tissue, testing for theeffects of various agents on the growth and differentiation of thetissue, as well as identification of agents involved with the growth anddifferentiation.

[0015] U.S. Pat. No. 5,753,505 issued May 19, 1998, provides an isolatedcellular composition comprising greater than about 90% mammalian,non-tumor-derived, neuronal progenitor cells which express aneuron-specific marker and which can give rise to progeny which candifferentiate into neuronal cells. Also provided are methods of treatingneuronal disorders utilizing this cellular composition.

[0016] U.S. Pat. No. 5,759,793 issued Aug. 6, 1996, provides a methodfor both the positive and negative selection of at least one mammaliancell population from a mixture of cell populations utilizing amagnetically stabilized fluidized bed. One application of this method isthe separation and purification of hematopoietic cells. Target cellpopulations include human stem cells.

[0017] U.S. Pat. No. 5,789,148 issued Aug. 4, 1998, discloses a kit,composition and method for cell separation. The kit includes acentrifugable container and an organosilanized silica particle-basedcell separation suspension suitable for density gradient separation,containing a polylactam and sterilized by treatment with ionizingradiation. The composition includes a silanized silica particle-basedsuspension for cell separation which contains at least 0.05% of apolylactam, and preferably treated by ionizing radiation. Also disclosedis a method of isolating rare blood cells from a blood cell mixture.

[0018] Within the past several years, mesenchymal stem cells (MSCs) havebeen explored as vehicles for both cell therapy and gene therapy. Thecells are relatively easy to isolate from the small aspirates of bonemarrow that can be obtained under local anesthesia: they are alsorelatively easy to expand in culture and to transfect with exogenousgenes. Prockop, D. J. Science 26: 71-74 (1997). Therefore, MSCs appearto have several advantages over hematopoietic stem cells (HMCs) for usein gene therapy. The isolation of adequate numbers of HSCs requireslarge volumes of marrow (I liter or more), and the cells are difficultto expand in culture. (Prockop, io D. J. (ibid.)).

[0019] There are several sources for bone marrow tissue, including thepatient's own bone marrow, that of blood relatives or others with MHCmatches and bone marrow banks. There are several patents that encompassthis source. U.S. Pat. No. 5,476,997 issued May 17, 1994, discloses amethod of producing human bone marrow equivalent. A human hematopoieticsystem is provided in an immunocompromised mammalian host, where thehematopoietic system is functional for extended periods of time. In thismethod, human fetal liver tissue and human fetal thymus are introducedinto a young immunocompromised mouse at a site supplied with a vascularsystem, whereby the fetal tissue results in formation of functionalhuman bone marrow tissue.

[0020] Human fetal tissue also represents a source of implantableneurons, but its use is quite controversial. U.S. Pat. No. 5,690,927issued Nov. 25, 1997, also utilizes human fetal tissue. Human fetalneuro-derived cell lines are implanted into host tissues. The methodsallow for treatment of a variety of neurological disorders and otherdiseases.

[0021] U.S. Pat. No. 5,753,491, issued May 19, 1998, discloses methodsfor treating a host by implanting genetically unrelated cells in thehost. More particularly, the present invention provides human fetalneuro-derived cell lines, and methods of treating a host by implantationof these immortalized human fetal neuro-derived cells into the host. Onesource is the mouse, which is included in the U.S. Pat. No. 5,580,777issued Dec. 3, 1996. This patent features a method for the in vitroproduction of lines of immortalized neural precursor cells, includingcell lines having neuronal and/or glial cell characteristics, comprisesthe step of infecting neuroepithelium or neural crest cells with aretroviral vector carrying a member of the myc family of oncogenes.

[0022] U.S. Pat. No. 5,753,506 issued May 19, 1998, reveals an in vitroprocedure by which a homogeneous population of multipotential precursorcells from mammalian embryonic neuroepithelium (CNS stem cells) isexpanded up to 10 fold in culture while maintaining their multipotentialcapacity to differentiate into neurons, oligodendrocytes, andastrocytes. Chemical conditions are presented for expanding a largenumber of neurons from the stem cells. In addition, four factors-PDGF,CNTF, LIF, and T3-have been identified which, individually, generatesignificantly higher proportions of neurons, astrocytes, oroligodendrocytes. These procedures are intended to permit a large-scalepreparation of the mammalian CNS stem cells, neurons, astrocytes, andoligodendrocytes. These cells are proposed as an important tool for manycell- and gene-based therapies for neurological disorders. Anothersource of stem cells is that of primate embryonic stem cells. U.S. Pat.No. 5,843,780 issued Dec. 1, 1998, utilizes these stem cells. A purifiedpreparation of stem cells is disclosed. This preparation ischaracterized by the following cell surface markers: SSEA-I (−); SSEA-3(+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In oneembodiment, the cells of the preparation have normal karyotypes andcontinue to proliferate in an undifferentiated state after continuousculture for eleven months. The embryonic stem cells lines are alsodescribed as retaining the ability to form trophoblasts and todifferentiate into tissues derived from all three embryonic germ layers(endoderm, mesoderm and ectoderm). A method for isolating a primateembryonic stem cell line is also disclosed in the patent.

[0023] There is substantial evidence in both animal models and humanpatients that neural transplantation is a scientifically feasible andclinically promising approach to the treatment of neurodegenerativediseases and stroke as well as for repair of traumatic injuries to brainand spinal cord. Nevertheless, alternative cell sources and novelstrategies for differentiation are needed to circumvent the numerousethical and technical constraints that now limit the widespread use ofneural transplantation. In short, there is a need for furtherdevelopment of readily available reliable sources of neural cells fortransplantation.

[0024] The use of umbilical cord blood for use in hematopoieticreconsitution has been around since the work of Ende in the early1970's. Because umbilical cord blood is rich in hematopoieticprecursors, including stem cells, it represents a good source of cellsfor hematopoietic reconstitution. To date, however, little work has beendone on using pluripotential stem cells or related neural precursorswhich are found in umbilical cord blood for neuronal transplantionperhaps because of the failure to realize the viable source of neuronalprecursors which can be found in umbilical cord blood.

[0025] Human cord and placental blood provides a rich source ofhematopoietic stem cells. On the basis of this finding, umbilical cordblood stem cells have been used to reconstitute hematopoiesis inchildren with malignant and nonmalignant diseases after treatment withmyeloablative doses of chemoradiotherapy. Sirchia and Rebulla, 1999Haematologica 84:738-47. Early results show that a single cord bloodsample provides enough hematopoietic stem cells to provide short- andlong-term engraftment, and that the incidence and severity ofgraft-versus-host disease has been low even in HLA-mismatchedtransplants. These results, together with our previous discovery thatbone marrow cells contain stem cells capable of differentiating intoneurons and glia, led to the present invention which uses cord blood ormononuclear cell fractions thereof to repair neuronal damage in brainand spinal cord. Sanchez-Ramos, et al. 1998. Movement Disorders 13(s2):122 and Sanchez-Ramos, et al., (2000) Exp. Neurol.

OBJECTS OF THE INVENTION

[0026] It is an object of the present invention to provide a novelsource of pluripotent stem and/or progenitor cells which can be readilydifferentiated into neuronal and glial cells to be used intransplantation in the brain and spinal cord of a patient and for thetreatment of neurodegenerative diseases.

[0027] It is an additional object of the invention to providepharmaceutical compositions comprising effective amounts orconcentrations of neural cells for use in transmplantation and methodsfor treating neurodegenerative diseases, or brain or spinal cordinjuries or damage.

[0028] It is another object of the invention to provide methods forisolating and inducing differentiation of pluripotent stem and/orprogenitor cells into neuronal and glial cells which can be used intranplantation procedures or for the treatment of neurodegenerativediseases.

[0029] It is a further object to provide a method of treatingneurodegenerative diseases and spinal cord/brain injury using neuraland/or neuronal and/or glial cells derived from umbilical cord blood.

[0030] It is yet a further object of the invention to provide a methodof transplanting neural and/or neuronal and/or glial cells derived fromumbilical cord blood in order to repair damaged organs of a patient'snervous system such as the brain and spinal cord.

[0031] These and/or other objects of the invention may be readilygleaned from the description of the invention which follows.

BRIEF DESCRIPTION OF THE INVENTION

[0032] The present invention relates to the unexpected discovery thatfresh or reconstituted umbilical cord blood or a mononuclear cellularfraction thereof is a novel source of cells which can be differentiatedinto neural cells and/or neuronal tissue and used for neuronaltransplantation (autografting as well as allografting), thus obviatingthe need to use pooled neuronal fetal tissue or bone marrow tissue,which is often hard to obtain. Thus, the present invention may be usedfor autografting (cells from an individual are used in that sameindividual), allografting (cells from one person are used in anotherperson) and xenografting (transplantation from one species to another).In this aspect of the present invention, it has unexpectedly beendiscovered that umbilical cord blood from neonates or fetuses comprisescells which may be induced to become neurons in vitro and in vivo. Thesecells may be used in autologous or allogeneic transplantation (grafting)procedures to improve neurological deficit and to effect transplantationand repair of neural/neuronal tissue, in particular, tissue of the brainand spinal cord and to treat neurodegenerative diseases of the brain andspinal cord.

[0033] In one aspect according to the present invention, umbilical cordblood derived neural cells are suitable for grafting into a patient'sbrain or spinal cord. These neural cells may be purified and/orincubated with a differentiation agent by any one or more of the methodsotherwise described in the present specification or alternatively, thesecells may be obtained from crude mononuclear cell fractions of umbilicalcord blood and used directly without further purification ordifferentiation. In other aspects, umbilical cord blood may be usedwithout further purification.

[0034] In another aspect of the present invention, there is presented amethod for obtaining neural cells from umbilical cord blood, the methodcomprising the steps of obtaining umbilical cord blood, selectingumbilical cord pluripotential stem cells or progenitor cells which areneural precursor cells and incubating the umbilical cord stem cells orprogenitor cells with a differentiation agent to change the phenotype ofthe cells to produce a population of neural cells which are capable ofbeing transplanted. The steps of the method may also be changed suchthat all of the cells (for example, from an umbilical blood sample or amononuclear cell fraction thereof) are incubated with a differentiationagent prior to separation of the neural phenotype cells.

[0035] The method of the present invention may include the step ofseparating the pluripotential stem and progenitor cells from apopulation of mononuclear cells obtained from umbilical cord blood usinga magnetic cell separator to separate out all cells which contain a CDmarker, and then expanding the cells which do not contain a marker in agrowth medium containing a differentiation agent such as retinoic acid,fetal neuronal cells or a growth factor such as BDNF, GDNF and NGF ormixtures, thereof, among numerous others. Preferably, a mixture ofretinoic acid and at least one growth factor, for example, nerve growthfactor, is used as the differentiation agent. The retinoic acid may be9-cis retinoic acid, all-transretinoic acid and mixtures thereof. Theseparation and incubation (differentiation) steps, may be interchanged.

[0036] Alternatively, an enriched cell population of pluripotent stemand/or progenitor cells may be obtained from a population of mononuclearcells obtained from umbilical cord blood by subjecting the mononuclearpopulation to an amount of an anti-proliferative agent (such as Ara-C[cytidine arabinoside] or methotrexate, among others) effective toeliminate all or substantially all proliferating cells and then exposingthe remaining non-proliferating cells to a mitogen such as epidermalgrowth factor or other mitogen (including other growth factors) toprovide a population of differentiated cells and quiescent cells(pluripotent stem or progenitor cells) which population is grown inculture medium such that the quiescent cells are concentrated in thecell population to greatly outnumber the differentiated cells. Thepluripotent stem and/or progenitor cells obtained may then be grown in acell medium containing a differentiation agent as generally describedabove in order to change the phenotype of the stem and/or progenitorcells to neuronal and/or glial cells which cells may be used intransplantation procedures directly without further purification.

[0037] The umbilical cord blood sample from which the pluripotent stemand/or progenitor cells are obtained may be fresh umbilical cord blood,reconstituted cryopreserved umbilical cord blood or a fresh orreconstituted cryopreserved mononuclear fraction thereof.

[0038] Novel compositions according to the present invention compriseumbilical cord blood or a mononuclear cellular fraction thereof, incombination with an effective amount of at least one neural celldifferentiation agent. Neural cell differentiation agents for use in thepresent invention include for example, retinoic acid, fetal or matureneuronal cells including mesencephalic or striatal cells or a growthfactor or cytokine such as brain derived neurotrophic factor (BDNF),glial derived neurotrophic factor (GDNF), glial growth factor (GFF) andnerve growth factor (NGF) or mixtures, thereof. Additionaldifferentiation agents include, for example, growth factors such asfibroblast growth factor (FGF), transforming growth factors (TGF),ciliary neurotrophic factor (CNTF), bone-morphogenetic proteins (BMP),leukemia inhibitory factor (LIF), glial growth factor (GGF), tumornecrosis factors (TNF), interferon, insulin-like growth factors (IGF),colony stimulating factors (CSF), KIT receptor stem cell factor(KIT-SCF) , interferon, triiodothyronine, thyroxine, erythropoietin,thrombopoietin, silencers, (including glial-cell missing, neuronrestrictive silencer factor), antioxidants such as vitamin E(tocopherol) and vitamin E esters, among others including lipoic acid,SHC (SRC-homology-2-domain-containing transforming protein),neuroproteins, proteoglycans, glycoproteins, neural adhesion molecules,and other cell-signalling molecules and mixtures, thereof.

[0039] Also presented is a cell line of pluripotent stem and/orprogenitor cells produced by any one or more of the above-describedmethods such that the cells have the ability to migrate and localize tospecific neuroanatomical regions where they differentiate into neuronalor glial cells typical of the region at the cite of transplantation andintegrate into the tissue in a characteristic tissue pattern.Pharmaceutical compositions utilizing these cells or other neural cellsare also an aspect of the present invention.

[0040] The present invention also is directed to a kit for neuronaltransplantation comprising a flask with dehydrated culture medium and apluripotent stem and/or progenitor cells and/or other neural cells.

[0041] The present invention is also directed to a method for treating aneurodegenerative disorder or a brain or spinal cord injury orneurological deficit comprising administering to (preferably,transplanting in) a patient suffering from such injury, aneurodegenerative disorder or neurlogical deficit an effective amount ofneural and/or neuronal and/or glial cells according to the presentinvention. Neurodegenerative disorders which can be treated using themethod according to the present invention include, for example,Parkinson's disease, Huntington's disease, multiple sclerosis (MS),Alzheimer's disease, Tay Sach's disease (beta hexosaminidasedeficiency), lysosomal storage disease, brain and/or spinal cord injuryoccurring due to ischemia, spinal cord and brain damage/injury, ataxiaand alcoholism, among others, including a number which are otherwisedescribed herein.

[0042] The present invention is also directed to a method of treatingneurological damage in the brain or spinal cord which occurs as aconsequence of genetic defect, physical injury, environmental insult ordamage from a stroke, heart attack or cardiovascular disease (most oftendue to ischemia) in a patient, the method comprising administering(including transplanting), an effective number or amount of neural cellsobtained from umbilical cord blood to said patient, including directlyinto the affected tissue of the patient's brain or spinal cord.Administering cells according to the present invention to a patient andallowing the cells to migrate to the appropriate cite within the centralnervous system is another aspect of the present invention.

[0043] A method of obtaining neural and/or neuronal and/or glial cellsfor autologous transplantation from an individual's own umbilical cordblood comprises the steps of 1) harvesting mononuclear cells from freshor cryopreserved umbilical cord blood or a cryopreserved mononuclearfraction of umbilical cord blood; 2) separating out the pluripotent stemcells and/or progenitor cells from the cord blood or mononuclearfraction; 3) incubating the stem cells and/or progenitor cells in amedium which includes an effective amount of a mitogen; and 4)incubating the stem and/or progenitor cells obtained from step 3 with adifferentiation agent.

BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1 is representative of results of gel studies indicating thepresence of mRNA from neuronal phenotypes.

[0045]FIG. 2 is representative of microscopic examination ofimmunostained cultures of cells which are tested for immunoreactivitywith antibodies to neuronal markers. Certain of the figures evidencethat the cells were immunoreactive with Mushashi-1 (FIG. 2A), β-tubulinIII (FIG. 2B) and GFAP, a marker of astrocytes, (FIG. 2E).

[0046]FIGS. 3A, 3B and 3C show the results of neurological functionrecovery in animals receiving a mononuclear fraction of human umbilicalcord blood 1 day after MCAo as evidenced by adhesive removal, rotarodand NSS tests.

[0047]FIGS. 4A, 4B and 4C show the results of neurological functionrecovery in animals receiving a mononuclear fraction of human umbilicalcord blood 7 days after MCAo as evidenced by adhesive removal, rotarodand NSS tests.

[0048]FIG. 5 shows the results of immunostaining of brain sections withMAB1281 and evidences that the highest concentrations of cord bloodcells migrate to the injured tissue.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The following definitions are used throughout the specificationto describe the present invention.

[0050] The term “patient” is used throughout the specification todescribe an animal, preferably a human, to whom treatment, includingprophylactic treatment, with the compositions according to the presentinvention, is provided. For treatment of those infections, conditions ordisease states which are specific for a specific animal such as a humanpatient, the term patient refers to that specific animal. The term“donor” is used to describe an individual (animal, including a human)who or which donates umbilical cord blood for use in a patient.

[0051] The term “umbilical cord blood” or “cord blood” is usedthroughout the specification to refer to blood obtained from a neonateor fetus, most preferably a neonate and preferably refers to blood whichis obtained from the umbilical cord or the placenta of newborns. The useof cord or placental blood as a source of mononuclear cells isadvantageous because it can be obtained relatively easily and withouttrauma to the donor. In contrast, the collection of bone marrow cellsfrom a donor is a traumatic experience. Cord blood cells can be used forautologous transplantation or allogenic transplantation, when and ifneeded. Cord blood is preferably obtained by direct drainage from thecord and/or by needle aspiration from the delivered placenta at the rootand at distended veins.

[0052] The term “effective amount” is used throughout the specificationto describe concentrations or amounts of components such asdifferentiation agents, mitogens, neural and/or neuronal or glial cells,or other agents which are effective for producing an intended resultincluding differentiating stem and/or progenitor cells into neural,neuronal and/or glial cells, or treating a neurodegenerative disease orother neurological condition including damage to the central nervoussystem of a patient, such as a stroke, heart attack or accident victimor for effecting a transplantation of those cells within the patient tobe treated. Compositions according to the present invention may be usedto effect a transplantation of the neural cells within the compositionto produce a favorable change in the brain or spinal cord, or in thedisease or condition treated, whether that change is an improvement(such as stopping or reversing the degeneration of a disease orcondition, reducing a neurological deficit or improving a neurologicalresponse) or a complete cure of the disease or condition treated.

[0053] The term “neural cells” are cells having at least an indicationof neuronal or glial phenotype, such as staining for one or moreneuronal or glial markers or which will differentiate into cellsexhibiting neuronal or glial markers. Examples of neuronal markers whichmay be used to identify neuronal cells according to the presentinvention include, for example, neuron-specific nuclear protein,tyrosine hydroxylase, microtubule associated protein, and calbindin,among others. The term neural cells also includes cells which are neuralprecursor cells, i.e., stem and/or progenitor cells which willdifferentiate into or become neural cells or cells which will ultimatelyexhibit neuronal or glial markers, such term including pluripotent stemand/or progenitor cells which ultimately differentiate into neuronaland/or glial cells. All of the above cells and their progeny areconstrued as neural cells for the purpose of the present invention.Neural stem cells are cells with the ability to proliferate, exhibitself-maintenance or renewal over the life-time of the organism and togenerate clonally related neural progeny. Neural stem cells give rise toneurons, astrocytes and oligodendrocytes during development and canreplace a number of neural cells in the adult brain. Neural stem cellsare neural cells for purposes of the present invention. The terms“neural cells” and “neuronal cells” are generally used interchangeablyin many aspects of the present invention. Preferred neural cells for usein certain aspects according to the present invention include thosecells which exhibit one or more of the neural/neuronal phenotypicmarkers such as Musashi-1, Nestin, NeuN, class III β-tubulin, GFAP,NF-L, NF-M, microtubule associated protein (MAP2), S100, CNPase,glypican (especially glypican 4), neuronal pentraxin II, neuronal PAS 1,neuronal growth associated protein 43, neurite outgrowth extensionprotein, vimentin, Hu, internexin, O4, myelin basic protein andpleiotrophin, among others.

[0054] The term “administration” or “administering” is used throughoutthe specification to describe the process by which neural cellsaccording to the present invention are delivered to a patient fortreatment purposes. Neural cells may be administered a number of waysincluding parenteral (such term referring to intravenous andintraarterial as well as other appropriate parenteral routes),intrathecal, intraventricular, intraparenchymal (including into thespinal cord, brainstem or motor cortex), intracisternal, intracranial,intrastriatal, and intranigral, among others which term allows neuralcells to migrate to the cite where needed. Neural cells may beadministered in the form of whole cord blood or a fraction thereof (suchterm including a mononuclear fraction thereof or a fraction of neuralcells, including a high concentration of neural cells). The compositionsaccording to the present invention may be used without treatment with adifferentiation agent (“untreated”, i.e., without further treatment inorder to promote differentiation of cells within the umbilical cordblood sample) or after treatment (“treated”) with a differentiationagent or other agent which causes certain pluripotential stem and/orprogenitor cells within the cord blood sample to differentiate intocells exhibiting neuronal and/or glial phenotype. Administration willoften depend upon the disease or condition treated and may preferably bevia a parenteral route, for example, intravenously, by administrationinto the cerebral spinal fluid or by direct administration into theaffected tissue in the brain. For example, in the case of Alzheimer'sdisease, Huntington's disease and Parkinson's disease, the preferredroute of administration will be a transplant directly into the striatum(caudate cutamen) or directly into the substantia nigra (Parkinson'sdisease). In the case of amyotrophic lateral sclerosis (Lou Gehrig'sdisease) and multiple sclerosis, the preferred administration is throughthe cerebrospinal fluid. In the case of lysosomal storage disease, thepreferred route of administration is via an intravenous route or throughthe cerebrospinal fluid. In the case of stroke, the preferred route ofadministration will depend upon where the stroke is, but will often bedirectly into the affected tissue (which may be readily determined usingMRI or other imaging techniques).

[0055] Each of these conditions, however, may be readily treated usingother routes of administration including, for example, an intravenous orintraarterial administration of whole umbilical cord blood or amononuclear cell fraction thereof to treat a condition or disease state.In the case of such treatment, however, and in particular, the treatmentof amyotrophic lateral sclerosis (Lou Gehrig's disease), treatment ofthe patient using parenteral (in particular, intravenous orintraarterial) administration of whole umbilical cord blood or amononuclear cellular fraction thereof or other routes of administrationwill be performed preferably in the absence of radiation or othertreatment such as chemotherapy (which are often used to eliminate bonemarrow cells or other tissue in the patient in order to impair, destroyand replace hematopoietic cells) before, during or after administrationof the umbilical cord blood or mononuclear cell fraction, thereof.

[0056] The terms “grafting” and “transplanting” and “graft” and“transplantation” are used throughout the specification synonymously todescribe the process by which neural and/or neuronal cells according tothe present invention are delivered to the site within the nervoussystem where the cells are intended to exhibit a favorable effect, suchas repairing damage to a patient's central nervous system, treating aneurodegenerative disease or treating the effects of nerve damage causedby stroke, cardiovascular disease, a heart attack or physical injury ortrauma or genetic damage or environmental insult to the brain and/orspinal cord, caused by, for example, an accident or other activity.Neural cells for use in the present invention may also be delivered in aremote area of the body by any mode of administration as describedabove, relying on cellular migration to the appropriate area in thecentral nervous system to effect transplantation.

[0057] The term “essentially” is used to describe a population of cellsor a method which is at least 95+% effective, more preferably at leastabout 98% effective and even more preferably at least 99% effective.Thus, a method which “essentially” eliminates a given cell population,eliminates at least about 95+% of the targeted cell population, mostpreferably at least about 99% of the cell population. Neural cellsaccording to the present invention, in certain preferred embodiments,are essentially free of hematopoietic cells (i.e., the CD34+ cellularcomponent of the mononuclear cell fragment).

[0058] The term “non-tumorigenic” refers to the fact that the cells donot give rise to a neoplasm or tumor. Stem and/or progenitor cells foruse in the present invention are generally free from neoplasia andcancer.

[0059] The term “cell medium” or “cell media” is used to describe acellular growth medium in which mononuclear cells and/or neural cellsare grown. Cellular media are well known in the art and comprise atleast a minimum essential medium plus optional agents such as growthfactors, glucose, non-essential amino acids, insulin, transferrin andother agents well known in the art. In certain preferred embodiments atleast one differentiation agent is added to the cell media in which amononuclear cell fraction is grown in order to promote differentiationof certain cells within the mononuclear fraction into neural cells.

[0060] In a preferred aspect of the present invention, mononuclear cellsgrown in standard cellular media (preferably, at least a minimumessential medium supplemented with non-essential amino acids, glutamine,mercaptoethanol and fetal bovine serum (FBS)) are grown in a “neuralproliferation medium” (i.e., a medium which efficiently grows neuralcells) followed by growth in a “differentiation medium”, generally,which is similar to the neural proliferation medium with the exceptionthat specific nerve differentiation agents are added to the medium andin certain cases, other growth factors are limited or removed). Aparticularly preferred neural proliferation medium is a media whichcontains DMEM/F12 1:1 cell media, supplemented with 0.6% glucose,insulin (25 μg/ml), transferrin (100 μg/ml), progesterone 20 nM,putrescine (60 μM, selenium chloride 30 nM, glutamine 2 mM, sodiumbicarbonate 3 mM, HEPES 5 mM, heparin 2 ug/ml and EGF 20 nm/ml, bFGF 20ng/ml. One of ordinary skill will readily recognize that any number ofcellular media may be used to grow mononuclear cell fractions ofumbilical cord blood or to provide appropriate neural proliferationmedia and/or differentiation media.

[0061] The term “separation” is used throughout the specification todescribe the process by which pluripotent stem and/or progenitor cellsare isolated from a mononuclear cell sample or a sample which containscells other than the desirable stem and/or progenitor cells, forexample, umbilical cord blood or other fragment.

[0062] The term “mitogen” is used throughout the specification todescribe an agent which is added to non-proliferating cells obtainedfrom a mononuclear cell sample in order to produce differentiated cellsand quiescent cells (pluripotent stem and/or progenitor cells). Amitogen is a transforming agent which induces mitosis in certain cellsother than pluripotent stem and/or progenitor cells obtained fromumbilical cord blood. Preferred mitogens for use in the presentinvention include epidermal growth factor (EGF), among other agents suchas the less preferred pokeweed mitogen, which also may be used to inducemitosis. Mitogens are also any one or a combination of a variety ofgrowth factors which have been shown to exert mitogenic actions onneural and mesenchymal precursors. These growth factors are: EpidermalGrowth Factor (EGF) family ligands (EGF, Transforming Growth Factor α,amphiregulin, betacellulin, heparin-binding EGF and Heregulin), basicFibroblastic growth factors (bFGF) and other members of its super-family(FGF1, FGF4), members of Platelet-Derived Growth Factor family (PDGF AA,AB, BB), Interleukins, and members of the Transforming Growth Factor βsuperfamily.

[0063] The term “antiproliferative agent” is sued throughout thespecificaiton to describe an agent which will prevent the proliferationof cells during methods according to the present invention which enrichpluripotent stem and/or progenitor cells. Exemplary antiproliferativeagents include, for example, Ara-C, methotrexate and otherantiproliferative agents. Preferred antiproliferative agents are thoseagents which limit or prevent the growth of proliferating cells withinan umbilical cord blood sample or mononuclear cell fraction thereof sothat quiescent stem and/or progenitor cells may be enriched.

[0064] The term “differentiation agent” or “neural differentiationagent” is used throughout the specification to describe agents which maybe added to cell culture (which term includes any cell culture mediumwhich may be used to grow neural cells according to the presentinvention) containing pluripotent stem and/or progenitor cells whichwill induce the cells to a neuronal or glial phenotype. Preferreddifferentiation agents for use in the present invention include, forexample, antioxidants, including retinoic acid, fetal or mature neuronalcells including mesencephalic or striatal cells or a growth factor orcytokine such as brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF) and nerve growth factor (NGF) or mixtures,thereof. Additional differentiation agents include, for example, growthfactors such as fibroblast growth factor (FGF), transforming growthfactors (TGF), ciliary neurotrophic factor (CNTF), bone-morphogeneticproteins (BMP), leukemia inhbitory factor (LIF), glial growth factor(GGF), tumor necrosis factors (TNF), interferon, insulin-like growthfactors (IGF), colony stimulating factors (CSF), KIT receptor stem cellfactor (KIT-SCF), interferon, triiodothyronine, thyroxine,erythropoietin, thrombopoietin, silencers, (including glial-cellmissing, neuron restrictive silencer factor), SHC(SRC-homology-2-domain-containing transforming protein), neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, and othercell-signalling molecules and mixtures, thereof. Differentiation agentswhich can be used in the present invention are detailed in“Marrow-mindedness: a perspective on neuropoiesis”, by Bjorn Scheffler,et al., TINS, 22, pp. 348-356 (1999), which is incorporated by referenceherein.

[0065] The term “neurodegenerative disease” is used throughout thespecification to describe a disease which is caused by damage to thecentral nervous system and which damage can be reduced and/or alleviatedthrough transplantation of neural cells according to the presentinvention to damaged areas of the brain and/or spinal cord of thepatient. Exemplary neurodegenerative diseases which may be treated usingthe neural cells and methods according to the present invention includefor example, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, RettSyndrome, lysosomal storage disease (“white matter disease” orglial/demyelination disease, as described, for example by Folkerth, J.Neuropath. Exp. Neuro., 58, Sep. 9, 1999), including Sanfilippo, TaySachs disease (beta hexosaminidase deficiency), other genetic diseases,multiple sclerosis, brain injury or trauma caused by ischemia,accidents, environmental insult, etc., spinal cord damage, ataxia andalcoholism. In addition, the present invention may be used to reduceand/or eliminate the effects on the central nervous system of a strokeor a heart attack in a patient, which is otherwise caused by lack ofblood flow or ischemia to a site in the brain of said patient or whichhas occurred from physical injury to the brain and/or spinal cord. Theterm neurodegenerative diseases also includes neurodevelopmentaldisorders including for example, autism and related neurologicaldiseases such as schizophrenia, among numerous others.

[0066] Selecting for umbilical cord pluripotential stem and/orprogenitor cells according to the present invention can be done in anumber of ways. For example, the cells may be selected using, forexample a magnetic cell separator (FACS) or other system which removesall cells which contain a CD marker and then the remaining cells may beexpanded in growth medium or differentiated in growth medium whichincludes a differentiation agent. Alternatively, an enriched populationof stem and/or progenitor cells may be obtained from a sample ofmononuclear cells by subjecting the cells to an agent such as Ara-C orother anti-proliferative agent such as methotrexate, which causes thedeath of proliferating cells within a sample (the stem and/or progenitorcells are non-proliferating and are unaffected by the agent). Theremaining cells may then be grown in a cell culture medium whichcontains a mitogen to produce a population of differentiated andquiescent cells, which cell population may be further grown toconcentrate the quiescent cells to the effective exclusion of thedifferentiated cells (the quiescent cells in the final cell medium willgreatly outnumber the original differentiated cells which do not grow inthe medium). The quiescent cells may then be induced to adopt a numberof different neural phenotypes, which cells may be used directly intransplantation.

[0067] Additional in vitro differentiation techniques can be adaptedthrough the use of various cell growth factors and co-culturingtechniques known in the art. Besides co-culturing with fetalmesencephalic or striatal cells, a variety of other cells can be used,including but not limited to accessory cells, and cells from otherportions of the fetal and mature central nervous system.

[0068] The term “gene therapy” is used throughout the specification todescribe the transfer and stable insertion of new genetic informationinto cells for the therapeutic treatment of diseases or disorders. Theforeign gene is transferred into a cell that proliferates to spread thenew gene throughout the cell population. Thus, stem cells, orpluripotent progenitor cells according to the present invention eitherprior to differentiation or preferably, after differentiation to aneural cell phenotype, are the target of gene transfer, since they areproliferative cells that produce various progeny lineages which willpotentially express the foreign gene.

[0069] The following written description provides exemplary methodologyand guidance for carrying out many of the varying aspects of the presentinvention.

[0070] General Methods

[0071] Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,New York (1989, 1992), and in Ausubel et al., Current Protocols InMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).Polymerase chain reaction (PCR) is carried out generally as in PCRProtocols: A Guide To Methods and Applications, Academic Press, SanDiego, Calif. (1990). Reactions and manipulations involving othernucleic acid techniques, unless stated otherwise, are performed asgenerally described in Sambrook, et al., 1989, Molecular Cloning: aLaboratory Manual, Cold Springs Harbor Laboratory Press, and methodologyas set forth in U.S. Pat. Nos. 4,666,828; 4,683,202, 4,801,531;5,192,659; and 5,272,057 and incorporated herein by reference. In-situPCR in combination with Flow Cytometry can be used for detection ofcells containing specific DNA and mRNA sequences (see, for example,Testoni et al. Blood 87:3822 (1996)).

[0072] Standard methods in immunology known in the art and notspecifically described are generally followed as in Stites et al. (eds),BASIC AND CLINICAL IMMUNOLOGY, 8'th Ed., Appleton & Lange, Norwalk,Conn. (1994); and Mishell and Shigi (eds), Selected Methods In CellularImmunology, W. H. Freeman and Co., New York (1980).

[0073] Immunoassays

[0074] In general, immunoassays are employed to assess a specimen suchas for cell surface markers or the like. Immunocytochemical assays arewell known to those skilled in the art. Both polyclonal and monoclonalantibodies can be used in the assays. Where appropriate otherimmunoassays, such as enzyme-linked immunosorbent assays (ELISAs) andradioimmunoassays (RIA), can be used as are known to those in the art.Available immunoassays are extensively described in the patent andscientific literature. See, for example, U.S. Pat. Nos. 3,791,932;3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262;3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 2o4,879,219; 5,011,771 and 5,281,521 as well as Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Springs Harbor, N.Y. (1989). Numerousother references also may be relied on for these teachings.

[0075] Antibody Production

[0076] Antibodies may be monoclonal, polyclonal or recombinant.Conveniently, the antibodies may be prepared against the immunogen orimmunogenic portion thereof, for example, a synthetic peptide based onthe sequence, or prepared recombinantly by cloning techniques or thenatural gene product and/or portions thereof may be isolated and used asthe immunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art asdescribed generally in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Springs Harbor, N.Y. (1988) andBorrebaeck, Antibody Engineering- A Practical Guide by W. H. Freeman andCo. (1992). Antibody fragments may also be prepared from the antibodiesand include Fab and F(ab′)2 by methods known to those skilled in theart. For producing polyclonal antibodies a host, such as a rabbit orgoat, is immunized with the immunogen or immunogenic fragment, generallywith an adjuvant and, if necessary, coupled to a carrier; antibodies tothe immunogen are collected from the serum. Further, the polyclonalantibody can be absorbed such that it is monospecific. That is, theserum can be exposed to related immunogens so that cross-reactiveantibodies are removed from the serum rendering it monospecific.

[0077] For producing monoclonal antibodies, an appropriate donor ishyperimmunized with the immunogen, generally a mouse, and splenicantibody-producing cells are isolated. These cells are fused to immortalcells, such as myeloma cells, to provide a fused cell hybrid that isimmortal and secretes the required antibody. The cells are thencultured, and the monoclonal antibodies harvested from the culturemedia.

[0078] For producing recombinant antibodies, messenger RNA fromantibody-producing B-lymphocytes of animals or hybridoma isreverse-transcribed to obtain complementary DNAs (cDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system.Antibody cDNA can also be obtained by screening pertinent expressionlibraries. The antibody can be bound to a solid support substrate orconjugated with a detectable moiety or be both bound and conjugated asis well known in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone & Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, Oxford,1982). The binding of antibodies to a solid support substrate is alsowell known in the art. (see for a general discussion Harlow & Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPublications, New York, 1988 and Borrebaeck, Antibody Engineering—APractical Guide, W.H. Freeman and Co., 1992). The detectable moietiescontemplated with the present invention can include, but are not limitedto, fluorescent, metallic, enzymatic and radioactive markers. Examplesinclude biotin, gold, ferritin. alkaline phosphates, galactosidase,peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴C, iodination andgreen fluorescent protein.

[0079] Gene Therapy

[0080] Gene therapy as used herein refers to the transfer of geneticmaterial (e.g., DNA or RNA) of interest into a host to treat or preventa genetic or acquired disease or condition. The genetic material ofinterest encodes a product (e.g., a protein. polypeptide. and peptide,functional RNA, antisense) whose in vivo production is desired. Forexample, the genetic material of interest encodes a hormone, receptor,enzyme, polypeptide or peptide of therapeutic value. Alternatively, thegenetic material of interest encodes a suicide gene. For a review see“Gene Therapy” in Advances In Pharmacology, Academic Press, San Diego,Calif., 1997.

[0081] Administration of Cells for Transplantation

[0082] The cells of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement, including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

[0083] In the method of the present invention, the cells of the presentinvention can be administered in various ways as would be appropriate toimplant in the central nervous system, including but not limited toparenteral, including intravenous and intraarterial administration,intrathecal administration, intraventricular administration,intraparenchymal, intracranial, intracisternal, intrastriatal, andintranigral administration. In addition, all of these routes ofadministration may be used to effect transplantation of neural cells inthe present invention.

[0084] Methods of treating a patient for a neurodegenerative disease orbrain and/or spinal cord damage caused by, for example, physical injuryor by ischemia caused by, a stroke, heart attack or cardiovasculardisease comprise administering neural cells to said patient in an amountsufficient to effect a neuronal transplantation. One of ordinary skillmay readily recognize that one may use treated (i.e., cells exposed toat least one differentiation agent) or untreated neural cells for suchmethods, including fresh umbilical cord blood or a mononuclear fractionthereof.

[0085] Pharmaceutical compositions comprising effective amounts oftreated neural cells are also contemplated by the present invention.These compositions comprise an effective number of treated neural and/orneuronal and/or glial cells, optionally in combination with apharmaceutically acceptable carrier, additive or excipient. In certainaspects of the present invention, cells are administered to the patientin need of a transplant in sterile saline. In other aspects of thepresent invention, the cells are administered in Hanks Balanced SaltSolution (HBSS) or Isolyte S, pH 7.4. Other approaches may also be used,including the use of serum free cellular media. Such compositions,therefore, comprise effective amounts or numbers of treated neural cellsin sterile saline. These may be obtained directly by using fresh orcryopreserved umbilical cord blood or alternatively, by separating outthe mononuclear cells (MNC) from the whole blood, using density gradientseparation methods, among others, which are well known in the art (onesuch approach is presented herein). The isolated MNC may be useddirectly for administration/transplantation or may be treated with atleast one differentiation agent and used without further purification orisolation of neural cells, or alternatively, after treatment with atleast one differentiation agent, the neural cells may be isolated andused. Intravenous or intraarterial administration of the cells insterile saline to the patient may be preferred in certain indications,whereas direct administration at the site of or in proximity to thediseased and/or damaged tissue may be preferred in other indications.

[0086] Pharmaceutical compositions according to the present inventionpreferably comprise an effective number within the range of about1.0×10⁴ mononuclear cells to about 5.0×10⁷ mononuclear cells, morepreferably about 1×10⁵ to about 9×10⁶ mononuclear cells, even morepreferably about 1×10⁶ to about 8×10⁶ cells generally in solution,optionally in combination with a pharmaceutically acceptable carrier,additive or excipient. Effective numbers of neural cells, either withina sample of mononuclear cells or as concentrated or isolated neuralcells, may range from as few as several hundred or fewer to severalmillion or more, preferably at least about one thousand cells withinthis range. In aspects of the present invention whereby the cells areinjected in proximity to the brain or spinal cord tissue to be treated,the number of cells may be reduced as compared to aspects of the presentinvention which rely on parenteral administration (including intravenousand/or intraarterial administration).

[0087] In using compositions according to the present invention, freshor cryopreserved umbilical cord blood, a mononuclear fraction thereof,or fractions wherein neural cells are isolated and/or concentrated(using FACS or other separation methods for isolating neural cells froma population of mononuclear cells) may be used without treatment with adifferentiation agent or with an effective amount of a differentiationagent prior to being used in a neuronal transplant. In one preferredaspect of the present invention, a mononuclear fraction of cord blood isexposed to effective amounts of at least one differentiation agent (incell media) for a period to effect differentiation of cord stem cellsinto cells which express a neuronal and/or glial phenotype and aftersuch period, these cells are then used to effect a transplant in apatient.

[0088] In aspects of the invention in which cord blood stem cells aredifferentiated, the use of standard media which has been supplementedwith at least one or more differentiation agent is preferred. Acombination of retinoic acid and nerve growth factor (NGF) in effectiveamounts in certain aspects of the present invention as thedifferentiation agent is preferred. In certain preferred aspects of thepresent invention, neural cells are prepared from cord blood stems cellsgrowing in standard growth media in a two step approach using neuralproliferation media followed by a differentiation media. In this aspectof the present invention, cells grown in standard cellular media(preferably, at least a minimum essential medium supplemented withnon-essential amino acids, glutamine, mercaptoethanol and fetal bovineserum (FBS)) are initially grown in a “neural proliferation medium”(i.e., a medium which efficiently grows neural cells) followed by growthin a “differentiation medium”) (generally, similar to the neuralproliferation medium with the exception that specific nervedifferentiation agents are added to the medium and in certain cases,other growth factors are limited or removed). A preferred neuralproliferation medium is a media which contains DMEM/F12 1:1 cell media,supplemented with 0.6% glucose, insulin (25 μg/ml), transferrin (100μg/ml), progesterone 20 nM, putrescine (60 μM, selenium chloride 30 nM,glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM, heparin 2 μg/ml andEGF 20 nm/ml, bFGF 20 ng/ml. One of ordinary skill will readilyrecognize that any number of cellular media may be used to provideappropriate neural proliferation medium and/or differentiation medium.

[0089] The following examples are provided to illustrate or exemplifycertain preferred embodiments of the present invention illustrative ofthe present invention but are not intended in any way to limit thepresent invention.

EXAMPLES

[0090] Preparation of Cellular Samples

[0091] Cryopreserved or fresh umbilical cord blood (from human or ratumbilical cord that remains attached to placenta after delivery) isharvested and processed by Ficoll centrifugation. This results in nearly100% recovery of mononuclear cells which can be a) grafted directly intoa region of injured brain (eg in a rat stroke model or model ofneurodegenerative disease or trauma model) b) processed intosub-populations based on surface markers or c) cryopreserved for lateruse. Initial experiments with umbilical cord blood utilize all of themononuclear cells collected, without separation of CD34+ cellularcomponents (hematopoietic stem cells). Other experiments utilize cordblood that is depleted of CD34+ cells as described below. Approximately100,000 to 90,000,000 (1×10⁵ to about 9×10⁷, preferably at least about1×10⁶) cord blood cells are injected into the hemisphere renderedischemic by acutely obstructing blood flow to cerebral cortex.Assessment of recovery of limb function in the rat model of stroke isperformed at 2 and 4 and 8 weeks after grafting.

[0092] Preparation of Cord Blood Devoid of Hematopoietic Stem Cells(CD34+)

[0093] Using a magnetic cell sorting kit (Milteny Biotec, Inc, AuburnTex.), cord cells are labeled with CD34+ microbeads which marks cellsthat express hematopoietic stem cell antigen (CD34 in human samples).The cord blood cells are passed through an MS+ column for selection ofCD34+ cells. Two hundred μL of CD34+ Multi-sort MicroBeads is added per10⁸ total cells, mixed and incubated for 15 min at 6-12° C. Cells arewashed by adding 5-10× the labeling volume of buffer, centrifuged for 10min at 200×g and supernatant is removed. The cell pellet is resuspendedin 500 μL buffer. The MS+/RS+ column is washed with 500 μL of buffer.The cell suspension is applied to the column and the “negative” cellspassed through. The negative cells contain all cells in the cord bloodexcept the hematopoietic CD34+ cells. The column is then rinsed with 500μL of buffer three times. These washing are added to the “negative” cellfraction, centrifuged for 10 min at 220×g and the supernatant removed.The cell pellet is resuspended in 500 μL of buffer and diluted 1:1 withDulbecco's Minimal Essential Media (DMEM, GIBCO/BRL) and 10% fetalbovine serum (FBS), centrifuged through a density gradient (Ficoll-PaquePlus, 1.077 g/ml, Pharmacia) for 30 min at 1,000×g. The supernatant andinterface are combined and diluted to approximately 20 ml with growthmedium and plated in polyethylene-imine coated plastic flasks.

[0094] Defining the Optimal Medium For Generating Cord Blood Clones

[0095] The cord blood is suspended in serum-free medium composed of a1:1 mixture of Dulbecco's Minimal Essential Media (DMEM) and F12nutrient (Life Technologies-BRL). Other samples of cord blood materialare suspended in DMEM+10% Fetal Bovine Serum (FBS). The defined culturemedium is composed of DMEM/F12 (1:1) including glucose, glutamine,sodium bicarbonate and HEPES buffer plus a defined hormone and saltmixture (see Daadi and Weiss, 1999). To identify the optimal celldensity for cell survival and growth, cells are plated at densitiesvarying from 50,000 to 3×10⁶ cell/ml in Coming T75 culture flasks in thedefined media together with a specific mitogenic growth factor(s) (seebelow for the mitogens used). Cell survival and proliferation aremonitored very closely by examining culture flasks, noting and countingclones that arise daily. After a fixed culture period of 10 days thetotal number of clones are counted in both serum free and serumsupplemented cultures and compared. This gave us the number of precursorcells able to generate clones and rate of proliferation. Then theseclones are harvested, dissociated and the total viable cells counted andreseeded for a second passage and so on for the future passages. Thislast count allowed determination of the rate of proliferation and thesize of clones generated under each condition.

[0096] Caveats: From our experience with neural stem cells, we havefound that exposure of cells to serum induces differentiation andinhibits proliferation of neural lineages. Lower cell density also doesnot favor cell proliferation. Therefore, it is likely that we willobserve variability in the rate of cell growth depending on the presenceand absence of serum, the cell density and mitogen used. The mediacomposition may not be optimal for cell survival, proliferation andenrichment for a neural cell population. We also see differences inmorphologies and antigens expressed by the cells under these twoseparate conditions. Therefore, before each passage we identify thetotal number of clones and cells and also the proportion of neurons,astrocytes and oligodendrocytes as well as hematopoietic cell lineages.These data will guide us to constantly improve our medium formula bytrying new media components and, if necessary, a very low percentage ofBovine Serum Albumin (BSA) or neural stem cell conditioned media.

[0097] Identification of Sub-populations of Cord Blood Cells Responsiveto Specific Mitogens and Which Express Specific Neural Markers

[0098] The cord blood suspension is plated at an optimal cell density incorning T75 culture flasks in the optimal medium (as described above).This medium is supplemented with 10 to 20 ng/ml of one or a combinationof a variety of growth factors that have been shown to exert mitogenicactions on neural and mesenchymal precursors. These growth factors are:Epidermal Growth Factor (EGF) family ligands (EGF, Transforming GrowthFactor α, amphiregulin, betacellulin, heparin-binding EGF andHeregulin), basic Fibroblastic growth factors (bFGF) and other membersof its super-family (FGF1, FGF4), members of Platelet-Derived GrowthFactor family (PDGF AA, AB, BB), Interleukins, and members of theTransforming Growth Factor β superfamily. After a period of 10 to 15days in vitro, the cells are harvested and then reseeded in fresh mediumcontaining growth factor(s). To identify their immature nature, some ofthese cells are plated on poly-L-ornithine-coated glass coverslips in24-well Nunclon culture dishes. After, a period of 30 minutes to 1 hourthese cells are fixed with 4% paraformaldehyde and stained with avariety of markers for immature cells such as Nestin, vimentin, the CDmarkers CD34 (marker of hematopoietic stem cell) and CD33 and dendriticcell markers. The rest of the cells are reseeded in growth medium(medium containing mitogens) for the next passage. For each growthfactor used cell survival and proliferation is closely monitored andclones that arise every day are counted and the total number of clonesformed after a fixed period is determined. We also determine theproportion of cell types generated under each mitogen, as describedbelow.

[0099] Different rates of cell proliferation and/or proportion of celltypes are generated depending on the growth factor used and itsconcentration. Neuronal differentiation efficacy and the number ofpassages able to be carried out under each mitogen is determined.Studies have shown that the combination of EGF and FGF is required toisolate and propagate human neural stem cell. Therefore we testcombinations of these growth factors and potentiated the mitogenicaction by adding a specific component (heparin when bFGF is used as themitogen).

[0100] Establishment of Multipotent Clonally Derived Sub-populations ofCord Blood Stem Cells.

[0101] Isolation of precursor sub-populations based on their response toepigenetic signals generate a homogeneous cell population that behave ina predictable and similar manner when transplanted in vivo or challengedwith a specific treatment in vitro. These clonal cell lines are goodcandidates for clinical-grade development. From the initial startinggrowth factor responsive population of stable human cord blood cells,monoclonal cell populations are established as previously described(Daadi and Weiss, J. Neurosci. 19, 11 4484, 1999). Clusters of cells aredissociated, counted and suspended in media-hormone mix at aconcentration of 1 cell per 15 μl and plated at 15 μl/well in Terasakimicrowells or a 96-well dish. Wells with single cells are immediatelyidentified and marked. Single cells are also randomly picked from thesuspension using a hand-pulled 10 μl micropipette and transferred into aTerasaki microwell containing 10-15 μl of media. Clonal development ismonitored once per day using the inverted microscope with phase-contrastoptics. Cultures are fed by replacing 2 μl with fresh medium every 2days.

[0102] Each single cell proliferates and generates a clone of cells. Afraction of these single founder cells have a slow growth rate or do notproliferate or even die after a few days in culture. From our experiencewith neural stem cells and using clonal cultures, some single cells mayundergo cell death because of the lack of neighboring cells that provideextracellular support for cell survival and in specific cases celldivision. If this is a problem, the founder cell are cultured inconditioned medium derived from bulk stem cell cultures, or in thepresence of the membrane extract of cord blood cells.

[0103] Characterization and Determination of the DifferentiationEfficacy of Each Clone.

[0104] In addition to growing a purified monoclonal human cordblood-derived stem cell populations, it is necessary to verify that eachgeneration of the clone exhibits all stem cell characteristics i.e.:ability to self renew, generate a large number of progeny and be able torespond to environmental cues and differentiate into different celltypes. These efficacy criteria are fundamental for the development andthe production of stable multipotent clones. Clonally derived cells (asdescribed above) are dissociated either by gentle mechanical triturationor using trypsin-EDTA. After the growth phase, part of the nextgeneration clone is cultured under differentiation conditions. Whencells grow as a cluster in suspension each clone is removed and platedin control media-hormone mixture without any mitogens on a glasscoverslip coated with an extracellular matrix (ECM). Different ECMsincluding laminin, Poly-L-ornithine and poly-D-lysine are tested fortheir potential differentiation effects. If cells grow as a monolayer,media containing the mitogen is removed by gentle suction and replacedby control fresh media (no mitogen). After a culture period of 10 to 15days, differentiated cells are fixed with paraformaldehyde and stainedfor various neural and, hematopoietic cell markers. Analysis of labeledsubpopulation are carried out using immunocytochemical techniques andFlow Cytometric Analysis. For neural lineages we use: anti-Nestin, andanti-Vimentin to label immature precursor cells; anti-Glial FibrillaryAcidic Protein to label Astrocytes, anti-O4, anti-Myelin Basic Proteinand anti-CNPase to identify oligodendrocytes, Anti-NeuN, Anti-β-tubulinclass III, Anti-Neuron Specific Enolase, Anti-human specificNeurofilament, Anti-MAP2 to identify neurons. Within this last neuronalpopulation we test for different neurotransmitter phenotype expressionlike GABA, Choline Acetyltransferase, Tyrosine Hydroxylase andSerotonin. We also test for hematopoietic cells: Some of thecharacterized multipotent stem cell clones are cryopreserved asdescribed in general methods section (see below) and the rest passagedand maintained in culture.

[0105] Cryopreservation

[0106] Clonally derived cord blood cells are resuspended in cellfreezing media comprising 10% dimethyl sulfoxide, 50% Fetal Bovine Serumand 40% of defined medium and stored under liquid nitrogen are wellknown in the art.

[0107] The mononucleoar layer from whole umbilical cord blood may beprepared for cryopreservation using the following methodology, whichsteps may be varied without significantly changing the cryopreservationoutcome.

[0108] Processing and Storage of Umbilical Cord Blood

[0109] 1. Sample Preparation

[0110] Anticoagulated cord blood is aliquotted into sterile 50 mlconical tubes and the volume measured accurately. A small sample isremoved for white cell count and sterility testing. A sample of plasmais removed at this time by centrifugation for cryopreservation. The cordblood is diluted 1:2 with sterile phosphate buffered saline (PBS) andmixed carefully to a maximum of 35 ml per tube.

[0111] Step 2: Density Gradient Separation

[0112] Mononuclear cells are obtained from the cord blood usingFicoll-Hypaque density centrifugation. Each tube of diluted cord bloodis underlayered with 10 ml of sterile Ficoll-Hypaque solution and thencentrifuged at 1200 g for 30 min. at room temperature. In thisprocedure, mononuclear cells containing progenitor cells (stem cells)form a layer at the Ficoll/plasma interface whereas red cells andgranular cells (granulocytes) pass through the gradient to the bottom ofthe tube. The mononuclear cells are removed carefully by aspiration.

[0113] STEP 3: Mononuclear cell preparation (MNC)

[0114] The mononuclear cells are collected in sterile 50 ml tubes anddiluted 1:2 with tissue culture medium (RPMI) and centrifuged at 1500 gfor 15 minutes. The cells are further washed in RPMI and resuspended toa fixed volume (14 ml) and a small sample removed for white cellenumeration and CD34+ cell determination.

[0115] STEP 4: Preparation of MNC for Cryopreservation

[0116] The cell suspension is then centrifuged at 1200 g for 10 mins andthe cells resuspended in 2.5 ml of RPMI. A small sample is removed forsterility testing. To this suspension. 2.5 ml of autologous plasmacontaining 10% dimethyl sulfoxide (DMSO) as cryoprotectant is addedslowly and the resulting suspension transferred to a labeled (bar coded)sterile, 5 ml cryovial.

[0117] STEP 5: Controlled rate freezing in liquid nitrogen

[0118] The samples are then cryopreserved using a controlled rate offreezing from 4 deg C. to −90 deg C. using the following protocol:

[0119] +4 degree C. to −3 degree C. at one degree C. per minute

[0120] −3 degree C. to −20 degree C. at 10 degree C. per minute

[0121] −20 degree C. to −40 degree C. at one degree C. per minute

[0122] −40 degree C. to −90 degree C. at 10 degree C. per minute.

[0123] The cryovials are then stored in vapor phase of liquid nitrogenat −196 degrees C.

[0124] Differentiating Culture Conditions

[0125] Three T75 flasks of 10-15 days old suspension cultures are spundown for 5 min at 400 rpm. The cells are removed and placed into a 12 mlcentrifuge tube and spun down for 5 min at 600 rpm. The growth medium isremoved, and cells resuspended in fresh control media (no EGF or othermitogen) plus hormone mix. This step is repeated one more time to ensurethe complete removal of the mitogen from the media. Dissociated cellsare plated in media hormone mix at a density of 1×10⁶ cells/ml onpoly-L-ornithine-coated (15 μg/ml; Sigma) glass coverslips in 24-wellNunclon culture dishes with 0.5 ml/well. After 7 to 14 days in culture,cells will change morphology into a neuron-like or glial-like phenotype.Following staining with specific antibodies which recognize markers ofneural precursors, of neurons and of glia, the differentiation efficacyof the hormone mix is quantified. The density of positively stained cellbodies are determined in at least 20 randomly selected fields from eachculture dish or well using the 40× objective. For quantitation of totalNeuN-ir, GFAP and nestin-ir cells produced, a total of 3 experiments areperformed resulting in total of 6 culture dishes or wells analyzed foreach condition. Neural differentiation efficacy of a growth factor (orhormone mixture) are calculated as the percentage of NeuN-immunoreactivecells (relative to total number of cells in a dish identified with DAPInuclear stain).

[0126] Indirect Immunocytochemistry

[0127] Rabbit polyclonal antisera and mouse monoclonal antibodiesdirected against specific antigens are used as primary antibodies forindirect immunofluorescence. Coverslips fixed with 4% paraformaldehydefor 20 min followed by three washes (10 min each) in phosphate buffersaline (PBS). After the PBS rinse, coverslips are processed for singleor dual labeling and incubated with primary antibodies generated fromdifferent species. The primary antibodies are made in PBS/10% normalgoat serum +0.3% triton X-100. After 2 hours incubation at 37° C., thecoverslip are rinsed in PBS. Fluorescent conjugated secondary antibodies(1:100, 1:200, Jackson ImmunoResearsh) are applied in PBS for 30 min atroom temperature. Coverslips are then washed three times (10 min each)in PBS, rinsed with water, placed on glass slides, and coverslippedusing Fluorsave (Calbiochem). Fluorescence is detected and photographedusing Zeiss Laser Scanning Confocal microscope (model LSM 510). Theprimary antibodies that are used are against: Nestin (1:1000;PharMingen), Vimentin (1:200, Boehringer), Glial Fibrillary AcidicProtein (1:500, Sigma), O4 (1:100, Chemicon), Myelin Basic Protein(1:200, Boehringer), and CNPase (1:500, Sternberger Monoclonals), NeuN(1:100, Chemicorp), β-tubulin class III (1:1000, Sigma), Neuron SpecificEnolase (1:100, Chemicon), human specific Neurofilament (1:150,Boehringer), MAP2 (1:200, Chemicon), GABA (1:5000, Sigma), CholineAcetyltransferase (1:200, Chemicon), Tyrosine Hydroxylase (1:4000,Incstar), Serotinin (1:200, Chemicon), type IV collagen (1:50, Dako),Laminin (1:300, Sigma), CD10 (1:100, PharMingen), muscle actin (1:1000,Sigma), HLA-DR (1:200, PharMingen), CD45 (1:100, PharMingen), Mac-1(1:100, Chemicon); alkaline phosphatase staining kit (Sigma #85L-2).

[0128] Flow Cytometric Analysis

[0129] To assess the actions of specific treatment on differentiation orto establish a relative profile within a culture condition of differentcell populations labeled with the markers mentioned above, the harvestedcells are subject to Flow Cytometric Analysis. Cells are rinsed withfluorescence activated cell sorting (FACS) buffer (EBSS and 1% HIFBS)and 1×10⁶ cells are added to 100 μl of FACS buffer supplemented with theappropriate primary antibodies and incubated at 4° C. for 30 min. Afterwashing, secondary antibodies are added and incubated at 4° C. for 30min. For biotinylated antibodies, isotope controls are used to setgates; otherwise, gates are set with cells alone. Cell viabilityismonitored using propidium iodide exclusion. Flow Cytometric Analysis isperformed with FACScan™ (Becton-Dickinson) with all events gated on theforward and side scatter.

[0130] Western Blotting

[0131] The culture is washed three times in cold phosphate bufferedsaline (PBS), scraped into ice-cold PBS, and lysed in ice-cold lysisbuffer containing 20 nM Tris/HCl (pH=8.0), 0.2 mM EDTA, 3% Nonidet P-40,2 mM orthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl,and 10 μg each of aprotinin and leupeptin per ml. After incubation onice for 10 min, the samples are centrifuged at 14,000×g for 15 min andsupernatants are collected. An aliquotis removed for total proteinestimation (bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sampleis separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody is blocked with 5% non-fat drymilk overnight at 4° C. Immunoblottingis carried out with theappropriate primary antibody followed by their corresponding peroxidaseconjugated secondary antibodies. The blots are developed by enhancedchemiluminescence method (ECL, Amersham).

[0132] Reverse Transcription-polymerase Chain Reaction (RT-PCR) andNorthern Analysis Total RNA is extracted using TRIzol (LifeTechnologies-BRL) according to the recommended protocol.

[0133] RT-PCR: aliquot of 1 μg of RNA is reverse-transcribed in thepresence of 50 mM Tris-HCL, pH 8.3, 75 mM KCL, 3 mM MgCl2, 10 mM DTT,0.5 mM dNTPs and 0.5 μg Oligo-dT (12-18) (Pharmacia) with 200 USuperscript Rnase H-Reverse Transcriptase (Life Technologies-BRL).Aliquots of cDNA equivalent to 40 ng of total RNA are amplified in 25 μlreactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 50pmol of each primer, 400 μM dNTPs, and 0.5 U AmpliTaq DNA polymerase(Perkin-Elmer). The PCR thermal profileis determined for each pair ofprimer sequence used.

[0134] Southern blot. 15 μl aliquots of the amplified PCR products arerun on a 2% agarose Tris-acetate gel containing 0.5 μg/ml ethidiumbromide. The bands are transferred by capillary action to a Hybond-N+membrane and detected using the appropriate radiolabeled probes. Theradioactive membrane is exposed overnight to a BioMaxMR autoradiographicfilm (Kodak) at −80° C.

[0135] Northern hybridization. Aliquots (20 μg) of total RNA arefractioned on agarose formaldehyde gels. The RNAis transferred bycapillary action from the gel matrix to Hybond-N+ (Amersham) using10×SSC, and fixed onto membrane by baking. These membranes arehybridized with the adequate radio-labeled probes, washed withdecreasing concentration of SSC, 0.1% SDS and exposed to a BioMaxMRautoradiographic film for 24 hours (Kodak) at −80° C.

[0136] Human Umbilical Cord Blood Contains Multi-Potent Progenitor CellsWhich Give Rise to Neural Lineage

[0137] General Methods Following the methods which are generally setforth above, cord blood was shown to contain cells which candifferentiate to neural cells.

[0138] Source of Cells

[0139] The mononuclear cells for this study were isolated from humanumbilical cord blood samples. The cord blood samples were obtained fromthe stump of the umbilical cord on the placental side postpartum.Between 50 and 100 ml of blood was obtained per procedure. Cells werespun down, resuspended in crypopreservative medium and frozen in liquidnitrogen until needed.

[0140] Handling of the Cells and Culture Media. The frozen cells werethawed, spun down, resuspended and plated in 75 mm culture flasks inminimal essential medium (DMEM) supplemented with 2 mM glutamine(100×stock from Gibco/BRL), 0.001% B-mercaptoethanol, 1× non-essential aminoacids (100× stock from Gibco/BRL) and 10% FBS (stem cell qualified,Gibco/BRL). After 24 to 72 hrs, the medium was replaced with serum free“Neural Proliferation Medium” which consisted of “N2” medium (DMEM/F121:1, Gibco/BRL) supplemented with 0.6% glucose, insulin 25 μg/ml,transferrin 100 ug/ml, progesterone 20 nM, putrescine 60 uM, seleniumchloride 30 nM, glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM,heparin 2 ug/ml and EGF 20 ng/ml, bFGF 20 ng/ml. Differentiation mediumconsisted of the “Neural Proliferation Medium” without the EGF and bFGF,but instead containing retinoic acid (0.5 μM) plus 100 ng/ml nervegrowth factor (NGF). Detection of proliferating cells was accomplishedby incubating cultures with bromodexoyuridine (BrdU) (5 μM) for 24-48hrs with subsequent visualization of BrdU immunoreactive cells.

[0141] Isolation of RNA. Total RNA was isolated from human cord bloodcells (or fractions thereof) using the RNA STAT-60 kit using theprotocol recommended by the manufacturer (Tel-Test “B”, Inc.Friendswood, Tex. 77546). Following RNA isolation, its OD density wasmeasured at 260 nm, and stored at −80° C. Integrity was tested on 1%non-denaturing Seakem LE agarose gel (FMC Bioproducts, Rockland, Me.).

[0142] DNA Microarray

[0143] Total RNA was prepared as above. Total RNA obtained from humancord blood cells, with or without RA+NGF treatment from differentbatches were pooled together for this experiment (15 to 20 g total RNAwas needed per chip). The human genome U95A array (HG-U95A) fromAffymetrix Inc. was used in this experiment. The single array represents˜12,500 sequences. The experiment was done at the Functional GenomicsCore, Microarray Facility at the H. Lee Moffitt Cancer Center & ResearchInstitute using GeneChip Fluidics station 400, a GeneChip Hybridizationoven, and an HP GeneArray™ scanner. Analysis of the GeneChip microarrayhybridization pattern was performed using GeneChip Analysis Suite 4.0software.

[0144] Reverse Transcription (RT). RT was performed using randomhexamers as primers. Final volume was 20 μl with 1 μg of total RNA fromeach fraction of cells. The reaction mixture contained 1 mM of eachdeoxynucleoside triphosphate (dNTP), 1 U/μl RNase inhibitor, 5 mM MgCl₂,2.5 U/μl Murine leukemia virus (MuLV) reverse transcriptase, 2.5 μMrandom hexamers in 50 mM KCl and 10 mM Tris-HCl (pH 8.3). It was firstincubated at room temperature for 10 min, and then at 42° C. for 15minutes. The mixture was then be heated at 99° C. for 5 minutes andcooled on ice for 5 min to inactivate the transcriptase.

[0145] Polymerase Chain Reaction (PCR). PCR was performed in the sametubes as RT, in 100 μl total volume. Final concentrations were 2 mMMgCl₂, 0.2 mM of each dNTP, and 2.5 U/100 μl Ampli Taq DNA polymerase inthe 50 mM KCl and 10 mM Tris-HCl buffer (pH 8.3). For generation ofvarious cDNA fragments, a PE 9700 thermocycler (Perkin Elmer, FosterCity, Calif.) was programmed as follows: 1 cycle at 95° C. for 105 sec,35 cycles at 95° C. for 15 sec, followed by 60° C. for 30 sec, andfinally 1 cycle at 72° C. for 7 min. Both RT and PCR was done usingPerkin Elmer's GeneAmp RNA PCR kit. To identify the presence of variousneuronal markers, primers were constructed based on published humansequences. For Nestin (accession #X65964), forward primer: nt 2524-2542and reverse primer: nt 2921-2903. For Musashi-1 (accession #AB012851),forward primer: nt 319-339 and reverse primer: nt 618-598. For Necdin(accession #AB007828), forward primer: nt 2374-2393 and reverse primer:nt 2767-2747. For Neurofilament subunit NF-L (accession #X05608),forward primer: nt 3155-3173 and reverse primer: nt 3521-3501. Primerswere selected using the SEQWEB (version 1.1) software available on theUSF computer network.

[0146] Antibodies

[0147] The primary antibodies used included: Musashi-1 (donated by Prof.H. Okano), Nestin (1:200, Chemicon); NeuN (1:100, Chemicon), class IIIβ-tubulin (1:200, Sigma); GFAP (1:500, Sigma), BrdU (1:400, Chemicon),MAP2 (1:200, Chemicon), pleiotrophin (1:400, R&D Systems).

[0148] Western Blot. Cultures were processed using standard methods forperformance of Western blot analysis using the following procedure.Cultures were washed three times in cold phosphate buffered saline(PBS), scraped into ice-cold PBS, and lysed in ice-cold lysis buffercontaining 20 nM Tris/HCl (pH 8.0), 0.2 mM EDTA, 3% Nonidet P-40, 2 mMorthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl, and10 μg each of aprotinin and leupeptin per ml. After incubation on icefor 10 min, the samples were centrifuiged at 14,000×g for 15 min andsupernatants were collected. An aliquot was removed for total proteinestimation (Bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample was separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody was blocked with 5% non-fat drymilk overnight at 4 C. The blots were analyzed using the Kodak DS 1DDigital Science Electrophoresis Documentation and Analysis System 120v.0.2.

[0149] Immunocytochemistry After 7-14 DIV, the cultures was fixed with4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 minutes. Thecultures were then washed 3 times with phosphate buffered saline priorto beginning immunocytochemistry.

[0150] Cell Counts For estimates of cell number in culture, 20 randomvisual fields (40× objective) in 4 culture dishes for each marker wereviewed. The total number of cells visualized under phase contrastmicroscopy and the number of positively labeled (immunoreactive) cellswas counted in each visual field. The mean number of labeled cells wasthen expressed as a percentage of the total number of cells per field.

[0151] Results

[0152] Cord blood cells, cultured in the presence and absence ofretinoic acid (RA) and Nerve Growth Factor (NGF), gave rise to cellsbearing neural progenitor markers as evidenced by profiles of gene andprotein expression. A total of 322 genes were either up- ordown-regulated by a factor of at least 2, evidenced by measurementsusing a human microarray “gene chip”. The greatest degree ofup-regulation (44 fold increase) was seen in the mRNA for neuriteoutgrowth extension protein or pleiotrophin. A significant degree ofdown regulation was seen in the expression of tenascin (decreased 8.8fold), an extracellular matrix protein that inhibits neurite outgrowthin developing neuronal tissues and in fibronectin (decreased 5.8 fold),an extracellular matrix protein that favors development of blood celllineages. Other transcripts associated with neurogenesis that increasedsignificantly (>2 fold) include glypican-4 (increased 4.9 fold),neuronal pentraxin II (increased 2.3 fold), neuronal growth associatedprotein 43 (increased 2.7 fold), neuronal PAS1 (increased 2.3 fold).Musashi-1 was upregulated 1.5 fold. A selection of other genesassociated with neurogenesis that were up- or down-regulated is listedin Table I. Concomitant with the increased expression of markersindicative of neurogenesis, there was a decrease in expression of genesassociated with hematopoiesis (Table II). The greatest changes occurredin the expression of HLA class I locus C heavy chain, macrophagereceptor MARCO, secreted T cell activation protein Attractin(attractin), leucocyte immunoglobulin-like receptor-8 (LIR-8), thymocyteantigen CD1c, erythropoietin receptor and erythropoietin.

[0153] In a parallel set of experiments, total RNA was extracted, andRT-PCR was performed. The mRNA for nestin and necdin was identified inboth control and RA+NGF treated cultures using primers based onpublished human sequences. In each case a product of appropriate lengthwas seen on the gel (FIG. 1A for untreated cells and FIG. 1B for treatedcelIs). Nestin is considered a marker of early neural development, butcan also be seen in endothelial precursors. Necdin is the gene thatcodes for neuron specific nuclear protein. The m-RNA for Musashi-1, theearliest marker of neural precursors was detected in RA+NGF treatedcultures and minimally detected in DMEM-treated controls. The mRNA forneurite outgrowth promoting protein (pleiotrophin) was detected inRA+NGF treated cells, but the signal was much weaker in untreatedcultures (FIG. 1C). The mRNA for glypican-4 was detected under bothconditions. The mRNA for GFAP, a marker of astroglial cells, was alsodetected under both conditions, though the signal was stronger in theRA+NGF treated cells. No messenger RNA for neurofilament subunit NF-Lwas detected in either treated or untreated cells although it was seento be up-regulated in the microarray. A negative RT control (withoutreverse transcriptase) was run with all the reactions to check forgenomic DNA contamination in the RNA preparation while human -actinprimers (Clontech) were used as a positive control. We also testedprimers for Musashi-1 and Neurofilament subunit NF-L using human brainRNA (Clontech) by RT-PCR; these each generated a single band ofappropriate length (data not shown).

[0154] Microscopic examination of immunostained cultures treated withRA+NGF revealed a heterogeneous mixture of cell types ranging from largeflat epithelioid cells to small spindle-shaped cells with fine branchingneuritic processes. A significant proportion (5-10%) of the small cellsin the RA+NGF treated cultures, but not control cultures treated withDMEM, were Musashi-1 immunoreactive (See FIG. 2A). A similar proportion(5%) of the small cells exhibited β-tubulin III immunoreactivity (FIG.2B). Approximately 50% of the cells were immunoreactive for BrdU,indicating that the cells were continuing to proliferate (data notshown). Antibodies to nestin (purchased from Chemicon, raised againstrat nestin) failed to recognize the human form of nestin, though theyhad been shown to react with rat nestin in rat bone marrow-derivedneural progenitor cells (Sanchez-Ramos, et al., Neuroscience News 3,32-43, 2000). Approximately 50% of RA+NGF treated cells wereimmunoreactive for GFAP, a marker of astrocytes (FIG. 2E). Western blotsof the cultures confirmed the presence of Musashi-1 protein,β-tubulin-III protein, pleiotrophin GFAP and NeuN in both treated anduntreated cells. Densitometric analysis of the blots showed that NGF+RAtreatment increased protein expression (relative to β-actin) ofMusashi-1, β-tubulin-III, pleiotrophin and NeuN (Table III). TABLE IExpression of Genes Associated with Neurogenesis Gene transcript Foldchange following RA + NGF Neurite outgrowth-promoting protein +44extracellular matrix-associated protein that enhances axonal growth inperinatal cerebral neurons [Raulo, 1992 #384] Glypican-4 +4.9 glypican-4is expressed in cells immunoreactive for nestin and the D1.1 antigen,markers of neural precursor cells. Glypican-4 expression not detected inearly postmitotic or fully differentiated neurons [Hagihara, 2000 #375]β-tubulin folding cofactor D ˜+4.6 Pro-galanin +3.9 Found in neurons ofarcuate nucleus of hypothalamus FE65 stat-like protein ˜+3.8 the exon9-inclusive (E9) form is exclusively expressed in neurons [Hu, 1999#369] Glial acidic fibrillary protein ˜+3.2 Neuron derived orphanreceptor ˜+2.2 Neuronal pentraxin II (NPTX2) ˜+2.3 member of a newfamily of proteins identified through interaction with a presynapticsnake venom toxin taipoxin; may function during synapse formation andremodeling [Kirkpatrick, 2000 #393] Neuronal growth protein 43 (GAP-43)˜+2.7 Identifies neurons, but also developing muscle cells [Moos, 1993#395] Neuronal PAS1 (NPAS1) +2.3 transcription factors selectivelyexpressed in the central nervous system [Zhou, 1997 #394] NeuronalDHP-sensitive, voltage-dependent, +2.1 calcium channel alpha-1D subunitBone morphogenetic protein 1 (BMP-1) +2 BMP-1/Tolloid is found at theneural plate/ectodermal transition. Expression is maintained in thepremigratory neural crest, and transiently in the migrating cephalicneural crest cells. [Marti, 2000 #391] Retinal glutamate transporterEAAT5 ˜+2 TrkC ˜+2 Receptor for neurotrophin-3 (NT3) ENO2 gene forneuron specific (gamma) +1.9 enolase Human brain protein recognized bythe sera of ˜+1.8 patients with paraneoplastic sensory neuronopathy Bonemorphogenetic protein 2A ˜+1.8 Neuronal PAS2 (NPAS2) +1.7 transcriptionfactors selectively expressed in the central nervous system [Zhou, 1997#394] Survival motor neuron pseudogene +1.7 Glial Growth Factor 2 +1.6Neural cell adhesion molecule (N-CAM) Exon ˜+1.6 SEC Follistatin-relatedprotein (FRP) +1.6 Microtubule-associated protein 2 (MAP2) ˜1.6Vesicular acetylcholine transporter +1.6 Neurofilament subunit M (NF-M)+1.5 Neurofilament subunit NF-L +1.5 Musashil ˜1.5 Bone morphogeneticprotein 11 (BMP11) +1.5 BMP-11 is expressed in the developing nervoussystem; at higher doses induces nervous tissue [Gamer, 1999 #392]Tenascin-C −8.8 Tenascin-C is an extracellular matrix protein thatinhibits neurite extension, and promotes cell proliferation andmigration [Thomas, 1996 #397; Anstrom, 1996 #398]

[0155] Table II Downregulation of Genes Associated with Development ofBlood Lines Fold decrease HLA class I locus C heavy chain −6.4Macrophage receptor MARCO −4.9 secreted T cell activation proteinAttractin −3.6 (attractin) alpha-1 collagen type II −3.0 Leucocyteimmunoglobulin-like receptor-8 (LIR- −2.8 8) Thymocyte antigen CD1c −2.5Erythropoietin receptor ˜−2.6 Erythropoietin ˜−2.4 Monocyte chemotacticprotein-2 −2.2 LAG-3 mRNA for CD4-related protein involved in ˜−2.3lymphocyte activation Interleukin-7 receptor (IL-7) −2.2 Complementreceptor type 1 −2.1 T cell receptor −2 p50-NF-kappa B homolog −2Lymphocyte-specific protein tyrosine kinase ˜−2 (LCK) LAG-3 mRNA forCD4-related protein involved in ˜−2.3 lymphocyte activation Erythrocytemembrane protein Rh30A (Rhesus ˜−2.1 antigen) Erythrocyte membraneprotein band 4.2 (EPB42) ˜−2.9 Leukocyte IgG receptor (Fc-gamma-R) −1.8Erythroblast macrophage protein EMP −1.5

[0156] TABLE III Densitometric Measurement of Expressed ProteinsSeparated by Western Blot Ratio to Ratio to MW β-Actin β-Actin Proteinmarker DMEM NGF % change Musashi-1 36 kD 0.805 0.93 +15.5% β-III tubulin75-80 kD 0.328 0.50 +52.4% Pleiotrophin 18 kD 0.179 0.315 +75.9% GFAP 46kD 0.538 0.515 −4.3% NeuN 51 kD 0.6 0.715 +19.1% β-Actin 42 kD

[0157] Discussion of Results

[0158] These findings demonstrate that human umbilical cord bloodcontains cells that can be induced to express markers of neuraldevelopment, including Mushashi-1, glypican-4 and β-tubulin III. Recentwork has demonstrated Musashi- 1 immunoreactivity in the developingand/or adult CNS tissues of frogs, birds, rodents, and humans (Kaneko,et al., Developmental Neuroscience 22, 139-53 (2000). The anti-Musashi-1monoclonal antibody has been shown to react with undifferentiated,proliferative cells of the sub-ventricular zone in the CNS of allvertebrates tested. Glypican-4, upregulated five-fold in the cord bloodcultures treated with RA+NGF, has been reported to be expressed in cellsimmunoreactive for nestin and the D1.1 antigen, other known markers ofneural precursor cells, but it has not been detected in earlypostmitotic or fully differentiated neurons (Hagihara, et al,Developmental Dynamics 219, 353-67 (2000). β-tubulin III is one of themost specialized tubulins specific for neurons (Fanarraga, et al..,European Journal of Neuroscience 11, 517-27 (1999). Both theupregulation and the post-translational processing of class-IIIbeta-tubulin are believed to be essential throughout neuronaldifferentiation (Laferriere, et al., Cell Motility & the Cytoskeleton35, 188-99 (1996) and Laferriere, et al., Biochemistry & Cell Biology75, 103-17 (1997).

[0159] The cord blood cultures treated with RA+NGF also increasedexpression of many genes specific for neurons including pentraxin II,GAP43, FE65 stat-like protein, neuronal PAS1 and PAS2. Neuronalpentraxin II is a member of a new family of proteins identified throughinteraction with a presynaptic snake venom toxin taipoxin. Neuronal-pentraxin-II may function during synapse formation and remodeling(Kirkpatrick, et al, Journal of Biological Chemistry 275, 17786-92(2000). Neuronal growth associated protein 43 (GAP43) is considered aspecific neuronal marker but may also be expressed in developingmyocytes (Moos, T. & Christensen, L. R. GAP43 identifies developingmuscle cells in human embryos. Neuroreport 4, 1299-302 (1993). FE65stat-like protein (the exon 9-inclusive form) is specifically expressedin neurons (Hu, et al., Journal of Neuroscience Research 58, 632-40(1999). Neuronal PAS1 and PAS2 are transcription factors selectivelyexpressed in the central nervous system (Zhou, et al.., Proceedings ofthe National Academy of Sciences of the United States of America 94,713-8 (1997). Other genes indicative of neurogenesis that were expressedfollowing treatment included the neurofilament subunits-NF-L and NF-M,microtubule associated protein 2 (MAP2), the vesicular acetyl cholinetransporter, and neuronal DHP-sensitive, voltage-dependent, calciumchannel alpha-1D subunit. Cord blood cells expressed mRNA for neuronalspecific enolase, but this protein is also expressed by many cells inbone marrow, especially megakaryocytes. The greatest change observed incord blood cultures treated with RA+NGF was a 44 fold increase inexpression of mRNA for an extracellular matrix-associated protein thatenhances axonal growth in perinatal cerebral neurons (Raulo, et al.,Journal of Biological Chemistry 267, 11408-16 (1992). At the same timethere was a significant decrease in expression of mRNA for tenascin, anextracellular matrix protein which inhibits neurite outgrowth (Kukekov,et al., Experimental Neurology 156, 333-44 (1999). There was alsoevidence for glial cell development. Increased expression of the glialcell marker GFAP was measured in the microchip data, and confirmed byimmunocytochemistry. Concomitant with the increased expression ofmarkers indicative of neurogenesis, there was a decrease in expressionof genes associated with development of blood cell lines.

[0160] The present findings provide evidence that cord blood contains amulti-potent cell capable of differentiating into a neural lineage. Theease with which the umbilical cord blood can be obtained, stored, andexpanded in culture could make this a preferable source of cells fortransplantation for neurodegenerative diseases, gene delivery to thecentral nervous system, and repair of brain and spinal cord injuries.

Example Identification and Isolation of Mononuclear Cells ExpressingNeuronal, Astrocytic or Oligodendrocytic Markers and Use of MononuclearCells to Effect Transplantation in Stroke

[0161] This series of experiments is directed to using both cell culturetechniques and an animal model of cerebral ischemia to establish humancord blood as a viable source of NSCs for the treatment of CNS diseaseor injury. These studies determine the existence of mononuclear cells incord blood that express neuronal, astrocytic or oligodendrocytic markersand identify those mononuclear cells that give rise to neural celllineages. The cord blood stem cells are shown to provide a stable,readily available source of NSCs which become functional neurons and arecapable of producing behavioral recovery at a comparable level to thatobserved with transplantation of fetal neurons.

[0162] General Methods.

[0163] Culture Media. The frozen cells are thawed, spun down andresuspended and plated in 75 mm culture flasks in minimal essentialmedium (DMEM) supplemented with 2 mM glutamine(100× stock fromGibco/BRL), 0.001% B-mercaptoethanol, 1× non-essential amino acids (100×stock from Gibco/BRL) and 10% FBS (stem cell qualified, Gibco/BRL).After 24 to 72 hrs, the medium is replaced with serum free “NeuralProliferation Medium” which consists of N2 medium (DMEM/F12 1:1,Gibco/BRL) supplemented with 0.6% glucose, insulin 25 ug/ml, transferrin100 ug/ml, progesterone 20 nM, putrescine 60 uM, selenium chloride 30nM, glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM, heparin 2 ug/mland EGF 20 ng/ml, bFGF 20 ng/ml. Differentiation medium consists of the“Neural Proliferation Medium” without the EGF and bFGF, but insteadcontaining retinoic acid (0.5 μM) plus a specific growth factor (NGF,BDNF, or GDNF)

[0164] Transfection of Cord Blood Cells With Fluorescent Green ProteinDriven By the Musashi-1 Promoter.

[0165] An ΔE1 adenovirus bearing hGFP under the control of theMushashi-1 promoter (AdP/Musashi) (generously donated by H. Okano ofJapan) are used to transfect umbilical cord blood cells. This adenoviralDNA vector is a plasmid DNA that contains a portion of the viral genomein which the E1 A region is deleted and the hGFP under control oftheMusashi-1 promoter has been inserted in the place of the E1A regionof the genome. Cells to be transfected are plated in 0.5 ml ofserum-free “Neural Proliferation Medium”. To each culture dish of cellsto be transfected 0.8 μg of the DNA is diluted and mixed into 50 μl ofOpti-Mem® I Reduced Serum Medium Without Serum (Life Technologies, Inc).Eight μl of Plus Reagent Mix is added and incubated at room temperaturefor 15 min. Lipofectin Reagent (Life Tech, Inc) is diluted and mixed ina second tube (0.5 μl into 50 μl of Opti-Mem I Reduced Serum MediumWithout Serum). After 30 min incubation at room temperature, theprecomplexed DNA is mixed with diluted Lipofectin Reagent and incubatedfor 15 min at room temperature. Then the DNAPlus-Lipofectin Reagentcomplexes (100 μl) are added to each well and mixed gently by rockingthe plate back and forth. The cultures are incubated at 37° C. in 5% CO2for 4-5 h. After 24 to 48 hrs, selected cultures are harvested to assessefficiency of transfection.

[0166] Isolation of RNA. Total RNA is isolated from human cord bloodcells (or fractions thereof) using the RNA STAT-60 kit using theprotocol recommended by the manufacturer (Tel-Test “B”, Inc.Friendswood, Tex. 77546). Following RNA isolation, its OD densityismeasured at 260 nm, and stored at −80° C. Integrityis tested on 1%non-denaturing Seakem LE agarose gel (FMC Bioproducts, Rockland, Me.).

[0167] Reverse Transcription (RT). RTis performed using random hexamersas primers. Final volumeis 20 μl with 1 μg of total RNA from eachfraction of cells. The reaction mixture contains 1 mM of eachdeoxynucleoside triphosphate (dNTP), 1 U/μl RNase inhibitor, 5 mM MgCl₂,2.5 U/μl Murine leukemia virus (MuL V) reverse transcriptase, 2.5 μMrandom hexamers in 50 mM KCl and 10 mM Tris-HCl (pH 8.3). It will firstbe incubated at room temperature for 10 min, and then at 42° C. for 15minutes. The mixture will then be heated at 99° C. for 5 minutes andcooled on ice for 5 min to inactivate the transcriptase.

[0168] Polymerase Chain Reaction (PCR). PCRis performed in the sametubes as RT, in 100 μl total volume. Final concentrations are 2 mMMgCl₂, 0.2 mM of each dNTP, and 2.5 U/100 μl Ampli Taq DNA polymerase inthe 50 mM KCl and 10 mM Tris-HCl buffer (pH 8.3). For generation ofvarious cDNA fragments, a PE 9700 thermocycler (Perkin Elmer, FosterCity, Calif.)is programmed as follows: 1 cycle at 95° C. for 105 sec, 35cycles at 95° C. for 15 sec, followed by 60° C. for 30 sec, and finally1 cycle at 72° C. for 7 min. Both RT and PCR are done using PerkinElmer's GeneAmp RNA PCR kit. To identify the presence of variousneuronal markers, primers are constructed based on published humansequences. For Nestin (accession #X65964), forward primer: nt 2524-2542and reverse primer: nt 2921-2903. For Musashi-1 (accession #AB012851),forward primer: nt 319-339 and reverse primer: nt 618-598. For Necdin(accession #AB007828), forward primer: nt 2374-2393 and reverse primer:nt 2767-2747. For Neurofilament subunit NF-L (accession #X05608),forward primer: nt 3155-3173 and reverse primer: nt 3521-3501. Primerswere selected using the SEQWEB (version 1.1) software available on theUSF computer network.

[0169] Western Blot. Cultures are washed three times in cold phosphatebuffered saline (PBS), scraped into ice-cold PBS, and lysed in ice-coldlysis buffer containing 20 nM Tris/HCl (pH=8.0), 0.2 mM EDTA, 3% NonidetP-40, 2 mM orthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mMNaCl, and 10 μg each of aprotinin and leupeptin per ml. After incubationon ice for 10 min, the samples are centrifuged at 14,000×g for 15 minand supernatants are collected. An aliquotis removed for total proteinestimation (Bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample is separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibodyis blocked with 5% non-fat drymilk overnight at 4 C.

[0170] MCAO Induction. Sprague Dawley rats are anesthetized withIsofluorane and an incision made from the caudal end of thesternomastoid and sternothyroid muscles extending toward the ears. Usingblunt dissection techniques, the right common carotid artery is exposedand carefully dissected free of the vagus nerve. The external carotidwill then be tied off and an embolus (a 40 cm length of 4.0monofilament)is inserted through the external carotid approximately 25mm into the internal carotid. At this point, the embolus is blocking theorigin of the right middle cerebral artery. The embolus is left in placefor 1 hr. After removal, the external carotid is cauterized and theincision closed. The animals are allowed to recover for 24 hr prior totransplantation.

[0171] Transplantation. The freshly isolated MNCs are resuspended inHBSS+15 mM HEPES at a cell concentration of 100,000 cells/μl. Thecoordinates for the injection site are 1.2 mm anterior and +2.7 mmlateral to the bregma and −5.2 and −4.7 mm ventral to the dura with thetoothbar set at zero. Five microliters of the cell suspension aredeposited at 2 sites in the striatum adjacent to the infarct site alonga single needle tract. Each injection of 2.5 μlis delivered at the rateof 1 μl/min. The needle is left in place for an additional 5 min afterthe injection and then withdrawn slowly. The incisionis closed withwound clips. For the transplantation of the expanded and/ordifferentiated MNCs, the cells are lifted from the culture flasks withgentle mechanical trituration or lifted with trypsin (0.25%) and 1 mMEDTA at 37 C. for 3-4 min and washed three times with HBSS+15 mM HEPES.Cell concentrationis adjusted to 100,000 cells/μl.

[0172] Behavioral testing methods. Twenty-four hours after stroke, theanimals undergo a standardized neurological screening exam measuring 5motor and postural activities to verify the extent of the MCAO damage.This battery is repeated at one month post stroke. In addition, theanimals are tested at both time points in the Passive Avoidance test oflearning and memory. In the acquisition phase of the test, the animal isplaced on a platform in the corner of a Plexiglas cage. When it stepsoff the platform, the rat will receive a scrambled foot shock(approximately 2 mA) for as long as it remains off the platform.Learning is measured by the amount of time required for the rat toremain on the platform continuously for 3 minutes, and the number oftimes it leaves the platform. Twenty-four hours later, retention ismeasured by placing the rat on the platform, and recording the latencyto step-down measured to a maximum of 3 min and the number ofstep-downs. Animals are also tested in the Rotorod Test of motorcoordination. The animal is placed on a revolving rod (16 rpm) and thelatency to the first fall as well as the number of falls in a 3 minutetest is recorded. The test is repeated twice for a total of 3 tests pertesting session with a minimum 30 min. rest between tests. The thirdbehavioral observation includes Spontaneous Activity Monitoring. Theanimals are placed in a square acrylic box overnight with an infraredgrid to measure movement and direction. The Elevated Body Swing Test, ameasure of motor asymmetry is also performed. The animal is held by thebase of the tail and lifted 2″ above the base of the cage. The directionthat the head and body is lifted is recorded. The test is repeated 20times. The final test is Skilled Forepaw Use. This is also a measure ofmotor asymmetry. The animal is placed in an acrylic chamber with twodescending staircases. Each step is baited with 5 food pellets. Thechamber is designed such that each staircase can only be reached by onepaw. The number of pellets retrieved measures the function of each limb.There is a 5 day training period, during which the animals are partiallyfood deprived. All behavioral data are reported as mean±sem.

[0173] Tissue Preparation in Culture Preparations. After 7-14 DIV, thecultures are fixed with 4% paraformaldehyde in 0.1 M phosphate buffer(PB) for 20 minutes. The cultures are then washed 3 times with phosphatebuffered saline prior to beginning immunocytochemistry.

[0174] Tissue Preparation of Brain Sections. The rats are sacrificedunder deep chloral hydrate (10%) anesthesia and transcardial perfusionof the brain with 50 ml of 0.1 M phosphate buffer (PB) and then 250 ml4% paraformaldehyde in 0.1 M PB performed. The brain is removed,post-fixed for 24 hr and cryopreserved in 20% sucrose prior to cutting30 μm thick frozen sections through the forebrain.

[0175] Immunohistochemistry. Single and double immunofluorescencehistochemistry are performed. Briefly, the floating sections will firstbe quenched by incubation in a 10% methanol, 3% hydrogen peroxidesolution in phosphate buffered saline (PBS) followed by pre-incubationin 10% normal serum (horse or goat; Vector) in 0.3% Triton-X100 (Sigma)in PBS. The sections are transferred to primary antibody in 2% normalserum, 0.3% Triton X-100/PBS and incubated overnight at 4° C. Theprimary antibodies that are used include: Musashi-1 (donated by Prof. H.Okano), Nestin (1:200, Chemi-Con); vimentin (1:500 Chemi-Con) as markersof early neural precursors; NeuN (1:100, Chemi-Con) and Hu (1:20Molecular Probes) class III β-tubulin (1:200, Sigma) to identify humanneurons at specific stages of development; human specific GFAP (1:200,Sternberger Monoclonals) to identify astrocytes; and O4 or 2′3′ cyclicnucleotide 3′ phosphodiesterase (CNPase, 1:200, Sigma) to identifyoligodendrocytes derived from the transplanted human MNCs. The sectionsare then washed in PBS before being placed in secondary antibodyconjugated to either fluorescein or rhodamine for 2 hours. The sectionsare rinsed in PBS, mounted and coverslipped with Vectashield.Confirmation that a cell is doubly-stained with be obtained byz-stacking analysis of images collected with a Zeiss ConfocalMicroscopes (LSM 510).

[0176] Cell Counts. For assessment of cell number in culture, 20 randomvisual fields (40× objective) in 4 culture dishes for each marker in 3replicates are viewed. The total number of cells and the number ofpositively labeled cells are counted. For each experimental condition,the number of positive cells and the total number of cell nuclei stainedwith 4′, 6-dimidinee-2′-phenylindole dihydrochloride (DAPI) aredetermined. The total counts are then expressed as a percentage of thetotal DAPI-stained nuclei. For quantification of immunofluorescence inbrain sections, an unbiased counting methodology are used. Neurons aredirectly counted in a small number of sections at predetermined uniformintervals for the entire set of sections containing specific CNS nuclei.Within each section to be counted the field of view is focused at thetop of the section using a 40× objective. The focus is then shiftedthrough the section and the number of positive profiles not present atthe top of the section is counted.

[0177] Analysis. The number of animals to be used in these studies wasbased on a power analysis of data obtained in previous experiments inthis laboratory. The analysis showed that a minimum of 10 animals pergroup is needed to find a difference in the variables of interest at asignificance level of p<0.05. All quantifiable results are expressed asmean±sem and are analyzed using Analysis of Variance (ANOVA). Allpost-hoc tests are conducted using a Scheffé test.

[0178] Identification and Isolation of Stem/progenitor Cells Present inUmbilical Cord Blood

[0179] The ideal way to identify and isolate neural progenitor cellsamong the heterogeneous population of mononuclear cord blood cells is toutilize a cell surface marker to which a fluorescent or magnetic beadantibody tag is attached to facilitate sorting and separation.

[0180] Unfortunately, both Nestin and Musashi-1 are located in thecytoplasm. Surface-based immunoselection strategies do not yet permitthe prospective identification or specific extraction of neuralstem/progenitor cells. A novel strategy has been used to identify andmonitor internal molecular markers of neural progenitor cells and toseparate the neural progenitors from other cells using fluorescenceactivated cell sorting (FACS) (N. S. Roy et al., Journal of NeuroscienceResearch 59, 321-31 (2000)). This method relies on coupling thepromoters required for neuroepithelial-specific gene expression with areporter gene (either lacZ or Green Fluorescent Protein-GFP). Morespecifically, cis-regulatory elements (the “promoter”) required for theexpression of Musashi-1 or α-tubulin-1 were placed upstream to thereporter gene GFP (Wang et al., Nature Biotechnology 16, 196-201 (1998)and N. S. Roy et al., Journal of Neuroscience Research 59, 321-31(2000)) Using this approach, neural progenitors and young neurons havebeen identified and selectively harvested from a variety ofheterogeneous samples, including both adult and fetal mammalianforebrains at different developmental stages (Wang, et al., supra, andKeyoung et al., Society for Neuroscience Abstract, 159 (2000)).

[0181] Experimental Design. We identify and separate neural progenitorcells by FACS of cord blood cells transfected with the gene for GFP,driven by the neuronal promoter α-tubulin-1 (Tα1) or by the Mushashi-1promoter. Mononuclear cells are obtained from the placental stump of theumbilical cord after delivery and processed by Ficoll centrifugation(See General Methods). This results in nearly 100% recovery ofmononuclear cells. These cells are cryopreserved in aliquots of 2million cells until they are to be used. After thawing, and plating inculture flasks in supplemented minimal essential medium (DMEM) plus FBS10% for 48 hrs, the medium is changed to “Neural ProgenitorProliferation Medium” for 2 days (See Methods for definition of themedia). Then, the mononuclear cells are transfected in a suspensionculture with a plasmid or viral vector containing the gene for GFP underthe control of P/Tα1 or Musashi-1 (See General Methods for details onTransfection technique and description of the vectors). After a 6-hourtransfection, the cells are spun down, resuspended in “Neural ProgenitorProliferation Medium” and plated in small culture flasks (See GeneralMethods). GFP should typically be expressed by appropriate target cellswithin 2 days of transfection. Flow cytometry and sorting of GFP+cellsare performed after 2-7 days in culture. Cells are washed, dissociatedand analyzed by light forward and right-angle (side) scatter, and forGFP fluorescence through a 510±20 nm band pass filter as they traversethe beam of the Laser (488 nm, 100 mW). Sorting is done using apurification-mode algorithm. Cells detected as being more fluorescentthan background are sorted at 1,00-3,000 cells/s. Sorted GFP+cells areplated in 24 well culture plates in “Neural Proliferation Medium” (SeeGeneral Methods for details) and BrdU. At 2 and 7 days post-FACS, thesorted cultures are fixed and immunostained for BrdU together witheither Mushashi-1, β-tubulin-III, Nestin, NeuN, MAP2, glial fibrillaryacidic protein (GFAP) or O4 (to detect oligodendrocytes).

[0182] Results and Alternative Method for Enriching Stem-like Cells.Cells transfected with the plasmid DNA encoding P/Tα1:GFP or the viralvector encoding P/Musashi:GFP identify neural progenitors and immatureneurons as evidenced by immunoreactivity for Mushashi-1, β-tubulin-III,and Nestin. In most instances, at least 50% of the Musashi-1(+) cellsare co-labeled with BrdU antibody indicating that the cells areproliferating. Based on the work of Wang et al., supra, manyMusashi1-driven GFP+cells should be labeled for up to 7 to 10 days.These neural progenitors and their daughters should be selected andsubstantially enriched by FACS. Further, they also showed that 0.36% ofadult ventricular zone dissociates expressed Tα1-driven GFP. Assumingthat only 0.01% of cord blood cells express Mushashi1-driven GFP, andthe transfection efficiency using the plasmid DNA is 12.5%, we estimatethat for every 5×10⁶ cord blood cells processed, we shall obtain625-1000 neural precursor cells. However, using an adenoviral vectorresults in a much greater transfection efficiency. For that reason wealso use a ΔE1 adenovirus bearing hGFP under the control of the Musashipromoter (AdP/Musash1:hGFP) provided generously by H. Okano of Japan.

[0183] In an alternative method, we first identify the least committedstem-like cells from the mononuclear cells in the cord blood and isolatethis population from both CD34+ and CD34− cells prior to inducingproliferation and promoter-based isolation of neural progenitors. Thebasic premise of this strategy is that stem cells are quiescent andexpress very few cell surface markers, except during a proliferationphase. Primitive stem cells fail to stain with Hoescht 33342 and PyroninY and can be separated on this basis using FACS. Further, separation ofcells that express P-gp, the transmembrane protein product of themultiple drug resistance gene (MDR), which is likely to be expressed incells that exhibit characteristics of stem cells, could also beperformed. These staining characteristics could be used to separate stemcells from cord blood mononuclear cells using FACS. If FACS demonstratesthat P-gp+ cells are also cells that fail to stain with the fluorescentdyes (Hoescht 33342 and Pyronin Y), then magnetic bead cell sorting areused to physically separate the P-gp immunoreactive cells from cordblood. In order to increase the yield of neural progenitor cells, it ispreferable to start with the smaller population of the least committedcells found in cord blood.

[0184] Assessing the Self-renewal Capacity of the Neural ProgenitorPopulation

[0185] In the example above, we identifiy a subpopulation of cellsenriched with neural progenitor cells or uncommitted stem cells. Itwill, therefore, be critical to expand the cell populations in order toobtain sufficient cells for study and eventually for transplantation. Inthis study we will determine whether there are differences in theability of the isolated populations of cells to proliferate and the bestagents for inducing proliferation in the neural progenitors.

[0186] Experimental Design. Mononuclear cells are obtained and thesubpopulations isolated as described above. We will focus primarily onthe GFP+ cells containing neural progenitors. These cells are plated inCorning T75 flasks with “Neural Progenitor Proliferation Medium”. Thisserum-free, defined medium contains epidermal growth factor (EGF) andbasic fibroblast growth factor (bFGF) and is used to induceproliferation of neural stem cells derived from fetal or adult brains.Once the cultures reach confluence (about 1 week), the cells are liftedby incubation with 0.25% trypsin, and 1 mM EDTA for 3-4 minutes. Analiquot of cells is replated with BrdU to assess the proportion of cellsthat are actively proliferating. The cells are replated after 1:3dilution with Neural Progenitor Proliferation medium. Cell yield andviability is also determined with the trypan blue dye exclusion assayafter each passage, for at least five passages.

[0187] Results. Neural stem cells proliferate with exposure to EGF orbFGF and the combination of these growth factors optimally allow for thecontinuous, rapid expansion and passaging of human neural stem cells.Alternatively, there is an extensive list of trophic factors andcytokines that may be more or less effective in inducing proliferation.These include other members of the EGF family such as transforminggrowth factor (TGF)-, amphiregulin, betacellulin and heregulin; FGF2 andthe related FGF1 and FGF4, platelet-derived growth factor family (PDGF),interleukins, and members of the TGF β superfamily. There may be somedegree of differentiation that occurs despite culturing in presence ofknown mitogens. The proportion of cells that continue to proliferate(determined by the BrdU assay before each passage) will guide us in theselection of the optimal mitogens for the neural progenitors.

[0188] Assessing the Capacity of Cord Blood Derived Neural Progenitorsto Differentiate into Meurons, Astrocytes or Oligodendrocytes in vitro.

[0189] The mononuclear cells which proliferate subpopulations ofmononuclear cells as established above can differentiate into neurons,astrocytes and oligodendrocytes. We have shown that a small populationof non-hematopoietic stem cells in the bone marrow stromal cell fractionwill differentiate into neurons and astrocytes. Moreover, preliminaryresults show that cord blood treated with RA+NGF for less than one weekexpress a marker seen in early neuronal development, β-tubulin-III. Inthis study, we demonstrate that treatment with “Differentiation Media”will drive our neural progenitors into neuronal and glial phenotypes.

[0190] Experimental Design. Mononuclear cells from the first or secondpassage of the subpopulations with the greatest proliferative capacityas determined above (GFP+cells) are replated in 35 mm culture dishes inthe presence of a “neuronal differentiation medium” (See Methods fordefinition) and a series of specific neurotrophic factors. The first tobe tried are brain derived neurotrophic factor (BDNF, 10 ng/ml) sincethis media has been used previously with bone marrow stromal cells todifferentiate the cells along neural lineages. After 7-14 days in vitro(DIV), cultures are processed for Western blotting, RT-PCR andimmunocytochemistry to identify cells that express neural markers. Themarkers to be examined include nestin, vimentin, glial fibrillary acidicprotein (GFAP) to label astrocytes, O4, myelin basic protein and CNPaseto identify oligodendrocytes and NeuN, β-tubulin class III, Hu,Neuron-Specific Nuclear antigen (NeuN), human specific neurofilament andmicrotubule associated protein (MAP-2) to identify neurons.Quantification is described in the General Methods.

[0191] Results. Neural markers are observed in the subpopulations chosenfor assay based on preliminary results with cord blood and resultsobtained with differentiation of bone marrow stromal cells. Further, thepopulation most likely to give rise to these neural cells is theGFP-expressing cells (driven by the Musashi-1 promoter). Alternatively,it may be necessary to use the stem-like cells obtained by selecting theleast committed of cells from the cord blood, nonetheless we are stillable to obtain significant numbers of differentiated neurons and gliafollowing treatment with differentiation media.

Example Expanded Population of Mononuclear Cells and Expression ofNeural Markers After Transplantation Into Middle Cerebral ArteryOcclusion (MCAO) Model of Stroke

[0192] In addition to demonstrating the existence of the neuralphenotype in vitro, it is important to show that the isolated anddifferentiated cells could express or maintain their neural phenotypeafter transplantation. The culture environment is easily controlled andmanipulated. The environment into which the cells are transplanted invivo is much less predictable; and there are many influences on thecells once they are placed in situ, some of which may alter theproliferative capacity or phenotypic lineage of the cell. Therefore, wedemonstrate that these stem-like cells maintain the ability to becomeneurons, astrocytes or oligodendrocytes in the brain.

[0193] Experimental Design. Sprague Dawley rats (n=10/group) areassigned to one of the following groups: 1) Middle cerebral arteryocclusion (MCAO); 2) MCAO with a striatal transplant of freshly isolatedmononuclear cells; 3) and 4) MCAO+expanded GFP+cells from the twosubpopulations with the highest proliferative capacity as determinedabove; 5) and 6) MCAO+expanded/minimally differentiated GFP+cells asdetermined above. The untreated cord blood cells in group 2 are labeledwith the fluorescent dye PKH26 for later identification in the brain andthen transplanted into the striatum in the penumbral region of theinfarct. (The isolated neural progenitors will already be labeled by theGFP). The animals are evaluated on a series of behavioral measures and aneurological exam at 24 hr and one month. This includes two paradigms wehave used to demonstrate behavioral deficits after stroke and recoveryfollowing transplantation, the passive avoidance test of cognitivefunction and the rotorod test of motor coordination. The animals willthen be perfused with 4% paraformaldehyde and the brains harvested forhistological and immunohistochemical analysis of graft survival andneural differentiation. Sections are examined for the presence ofPKH26-positive (or GFP+) cells, and cells that express human NuclearMatrix Antigen (NuMA), allowing a second method of identifying humancord blood-derived cells in the rat brain). Other sections aredouble-labeled for PKH26 or NuMA and Hu, class III β-tubulin, NeuN,GFAP, O4. The proportion (%) of cord blood derived cells (NuMA-ir andPKH26+) that express neuronal markers (NeuN, Hu, or class IIIβ-tubulin), glial markers (GFAP) and oligodendrocyte markers (O4) aredetermined.

[0194] Results. Based on our preliminary results we see both behavioralimprovements and surviving stem-cell progeny in the MCAO-injured brain.Behavioral improvements are observed in all transplant groups, althoughit is better in the expanded/minimally differentiated cells where theexpanded cell population have already been committed to a neural lineageand therefore may be expected to express more neurons. With the wholeMNC cell fraction, there are fewer of the subpopulation cells that giverise to neural cells than in the expanded subpopulations.

Example Effect of Transplanation of Human Umbilical Cord MononuclearCells in Stroke

[0195] In a small series of pilot studies, the cord blood mononuclearcells were transplanted into the striatum of animals that had eitherundergone a permanent or temporary (1 hr) middle cerebral arteryocclusion (MCAO). The cells (500,000 cells/implant) were transplantedimmediately upon thawing or were treated in culture for a week withvarious trophic factors (BDNF, NGF, EGF+bFGF) prior to transplantation.Preliminary results obtained from the temporary stroke model revealeddifferences between the groups on the rotorod test of motorcoordination. Animals which received the retinoic acid+NGF treatedmononuclear cells were able to stay on a rotating axle longer and felloff fewer times in the 3 minute test period than did all other animalsin the study. This study evidenced that the umbilical blood cellsprovide a novel cell source for transplantation in stroke which canimprove function.

Example Parenteral Administration of Cord Blood Fractions in theTreatment of Neurological Damage From Ischemia (Stroke)

[0196] Methods and Materials

[0197] 1. HUCB Sources and Preparation:

[0198] HUCB was provided and analyzed by Cryocell international, INC.The cells contain 77.2% 95% CD34+ cells, respectively. The specimen wasstored in liquid nitrogen and the cells were restored at 37° C. Aftercentrifugation at 1000 rpm/min for 5 min at 4° C., the cells were washedwith 0.1 M PBS. Nucleated HUCB were counted using a cytometer to ensureadequate cell number for transplantation. The final dilution isapproximately 3×10⁶ HUCB in 500 μl saline for injection in each rat.

[0199] 2. Animal MCAo Model:

[0200] Adult male Wistar rats (n=38) weighing 270-300 g were employed inall experiments. Briefly, rats were initially anesthetized with 3.5%halothane and maintained with 1.0-2.0% halothane in 70% N₂O and 30% O₂using a face mask. Rectal temperature was maintained at 37° C.throughout the surgical procedure using a feedback-regulated waterheating system. Transcient MCAo was induced using a method ofintraluminal vascular occlusion modified in our laboratory [Chen, etal., J Cereb Blood Flow Metab 1992;12(4): 621-628]. The right commoncarotid artery, external carotid artery (ECA) and internal carotidartery (ICA) were exposed. A length of 4-0 monofilament nylon suture(18.5-19.5 mm), determined by the animal weight, with its tip rounded byheating near a flame, was advanced from the ECA into the lumen of theICA until it blocked the origin of the MCA. Two hours after MCAo,animals were reanaesthetized with halothane and reperfusion wasperformed by withdrawal of the suture until the tip cleared the lumen ofthe ECA.

[0201] 3. In vitro-Chemotaxis Assay

[0202] 1) Ischemia Brain Tissue Extracts:

[0203] Animals were sacrificed at 6h, 24h and 1w (n=3 per time point)after the onset of MCAo; a normal control group (n=3) was employed inwhich the animals were not subjected to surgical procedures. Tissueextracts were obtained from the experimental rats and control rats.Forebrain tissues were immediately obtained from interaural 12 mm tointeraural 2 mm [Paxinos et al, The Rat Brain in StereotaxicCoordinates. Academic Press, San Diego. 1986]. Each specimen wasdissected on a bed of ice into hemispheres ipsilateral right side andcontralateral to the MCAo. The tissue sections were homogenized byadding IMDM (150 mg tissue/ml IMDM) and incubated on ice 10 min. Thehomogenate was centrifuged at 100,000 g for 20 min at 4° C. and thesupernatant extracted.

[0204] 2) Ischemia Brain Tissue Extract Assay on HUCB Migration

[0205] Chemotactic activity of ischemia brain tissue extracts towardHUCB at different time points was evaluated by using 48-well microchemotaxis chamber technique, as described [Xu et al, Hematology,4:345-356, 1999] with some modification. HUCBs were resuspended in IMDM(serum free) at 10⁶ cells/ml. Twenty-five microliters of tissue extractsprepared from normal and ischemic brain at 6h, 24h and 1w after MCAo(150 mg tissue/ml IMDM) were placed in the lower chamber of the 48-wellmicro chemotaxis chamber. A polycarbonate membrane (8 μm pore size)strip was place over the lower wells and 50μl of HUCB suspension (1×10⁶cells/ml) was place in each of the upper wells. Migration of HUCBs wasallowed for 5h at 37° C. incubation and the number of migrated cellsinto the lower wells was then measured.

[0206] 4. In vivo-treatment With HUCB:

[0207] Experimental groups: Group 1 (Control): MCAo alone without donorcell administration (n=10); Group 2: 3×10⁶ human UCB cells injectedintravenously at 24 h after MCAo (n=6); The animals of group 1, 2 weresacrificed at 14 days after MCAo. In order to test the effects ofdelayed (7 day) treatment, we included two additional groups. Group 3(Control): MCAo alone without donor cell administration (n=5) and ratswere sacrificed at 35 days after MCAo; Group 4: 3×10⁶ HUCB cells wereinjected intravenously at 7 days after MCAo and rats were sacrificed at35 days after MCAo (n=5).

[0208] Implantation procedures: At 1 or 7 days post-ischemia, randomlyselected animals received HUCB. Animals were anesthetized with 3.5%halothane and then maintained with 1.0-2.0% halothane in 70% N₂O and 30%O₂ using a face mask mounted in a Kopf stereotaxic frame. Approximately,3×10⁶ HUCB cells in 0.5 ml total fluid volume were injected into a tailvein.

[0209] Functional tests: In all animals, a battery of behavioral testswere performed before MCAo, and at 1, 7, 14, 21, 28, 35 days after MCAoby an investigator who was blinded to the experimental groups. Thebattery of tests consisted of:

[0210] 1) Rotarod test: An accelerating rotarod was used to measure ratmotor function [Hamm R. J., J Neurotrauma 11(2): 187-196 1994; and Chen,J Med; 31(1-2):21-30, 2000]. The rats were placed on the rotarodcylinder and the time the animals remained on the rotarod was measured.The speed was slowly increased from 4 rpm to 40 rpm within 5 min. Atrial ended if the animal fell off the rungs or gripped the device andspun around for two consecutive revolutions without attempting to walkon the rungs. The animals were trained 3 days before MCAo. The meanduration (in seconds) on the device was recorded with 3 rotarodmeasurements one day before surgery. Motor test data are presented aspercentage of mean duration (three trials) on the rotarod compared withthe internal baseline control (before surgery).

[0211] 2) Adhesive-removal somatosensory test [Schallert, Brain Res379(1): 104-111 1986; Hernandez, Exp Neurol, 102(3): 318-324 1988;Zhang, Neurol Sci, 174(2): 141-146, 2000; and Chen, Neuropharmacology,39(5): 711-716 2000]. Somatosensory deficit was measured both pre- andpostoperatively. All rats were familiarized with the testingenvironment. In the initial test, two small pieces of adhesive-backedpaper dots (of equal size, 113.1 mm²) were used as bilateral tactilestimuli occupying the distal-radial region on the wrist of eachforelimb. The rat was then returned to its cage. The time to remove eachstimulus from forelimbs was recorded on 5 trials per day. Individualtrials were separated by at least 5 min. Before surgery, the animalswere trained for 3 days. Once the rats were able to remove the dotswithin 10 seconds, they were subjected to MCAo.

[0212] 3) Modified Neurological severity score (mNSS): [Borlongan, BrainRes; 676(1): 231-234 1995; Shohami, Brain Res, 674(1): 55-62, 1995;Chen, Neurotrauma. 1996; 13(10):557-568 1996; Shaller Adv Neurol,73:229-238, 1997]. Neurological function was graded on a scale of 0 to18 (normal score 0; maximal deficit score 18). mNSS is a composite ofmotor, sensory, reflex and balance tests [Germano, J Neurotrauma;11(3):345-353 1994]. In the severity scores of injury, one score pointis awarded for the inability to perform the test or for the lack of atested reflex; thus, the higher score, the more severe is the injury.

[0213] 5. Histological and Immunohistochemical Assessment:

[0214] Animals were allowed to survive for 14 or 35 days after MCAo, andat that time animals were reanaesthetized with ketamine (44 mg/kg) andxylazine (13 mg/kg). Rat brains were fixed by transcardial perfusionwith saline, followed by perfusion and immersion in 4% paraformadehyde,and the brain, heart, liver, spleen, lung, kidney and muscle wereembedded in paraffin. The cerebral tissues were cut into seven equallyspaced (2 mm) coronal blocks. A series of adjacent 6 μm-thick sectionswere cut from each block in the coronal plane and were stained withhematoxylin and eosin (H&E). The seven brain sections were traced usingthe Global Lab Image analysis system (Data Translation, Malboro, Mass.).The indirect lesion area, in which the intact area of the ipsilateralhemisphere was subtracted from the area of the contralateral hemisphere,was calculated [Swanson, J. Cereb Blood Flow Metab, 10(2): 290-293,1990]. Lesion volume is presented as a volume percentage of the lesioncompared to the contralateral hemisphere.

[0215] Single and double immunohistochemical staining [Li, Brain Res,838(1-2): 1-10, 1999] was used to identify cells derived from HUCB.Briefly, a standard paraffin block was obtained from the center of thelesion, corresponding to coronal coordinates for bregma −1˜1 mm. Aseries of 6 μm thick sections at various levels (100 μm interval) werecut from this block and were analyzed using light and fluorescentmicroscopy (Olympus, BH-2). To detect the distribution of transplantedHUCB cells in other organs (i.e. heart, liver, lung, spleen, kidney andmuscle, bone marrow), 3 sections (6 μm thick, 100 μm interval) from eachorgan were obtained and numbers MAB1281 reactive cells measured. MAB1281(Mouse Anti-human nuclei monoclonal antibody, Chemicon International,Inc) is markers for human [Vescovi, et al., Exp Neurol; 156(1):71-831999]. After deparaffinization, sections were placed in boiled citratebuffer (pH 6.0) within a microwave oven (650-720W). After blocking innormal serum, sections were treated with the monoclonal antibody (mAb)against MAB 1281 diluted at 1:300 in PBS with FITC staining foridentification HUCB. Analysis of MAB1281 positive cells is based on theevaluation of an average of 10 histology slides of brain, 3 slides fromeach organ per experimental animal.

[0216] To visualize the cellular co-localization of MAB1281 andcell-type-specific markers in the same cells, fluorescein isothiocyanateconjugated antibody (FITC, Calbiochem, Calif. and red cyanine-5.18) wasemployed for double-label immunoreactivity. Each coronal section wasfirst treated with the primary MAb1281 mAb with FITC staining foridentification HUCB. As described above, and were followed withcell-type-specific antibodies, a neuronal nuclear antigen (NeuN forneurons, dilution 1:200; Chemicon, Calif.), microtubule associatedprotein 2 (MAP-2 for neurons, dilution 1:200; Boehringer Mannheim) andglial fibrillary acidic protein (GFAP for astrocytes, dilution 1:1000;Dako, Calif.) and FVIII (Von Willebrand Factor, dilution: 1:400;Dako)with CY5 staining. Negative control sections from each animalreceived identical preparations for immunohistochemical staining, exceptthat primary antibodies were omitted. A total of 500 MAb1281 positivecells per animal were counted to obtain the percentage of MAb1281 cellscolocalized with cell type specific markers (MAP-2, NeuN, GFAP, FVIII)by double staining.

[0217] Laser Scanning Confocal Microscopy (LSCM): Colocalization ofMAB1281 with neuronal (NeuN, MAP-2, GFAP) and endothelial cell (FVIII)markers were conducted by LSCM using a Bio-Rad MRC 1024 (argon andkrypton) laser-scanning confocal imaging system mounted onto a Zeissmicroscope (Bio-Rad, Cambridge, Mass.) [Zhang ZG, 1999] Forimmunofuorescence double-labbeled coronal sections, green (FITC forHUCB) and Red cyanine-5.18 (Cy5 for MAP-2, NeuN or GFAP) fluorochromeson the sections were excited by a laser beam at 488 nm and 647 nm;emissions were sequentially acquired with two separate photomultipliertubes through 522 nm and 680 nm emission filters, respectively. Areas ofinterest were scanned with a 40× oil immersion objective lens in260.6×260.6 m format in the x-y direction and 0.5 m in z direction.

[0218] 6. Statistical Analysis:

[0219] The behavior scores (rotarod test, adhesive-removal test and NSS)were evaluated for normality. Repeated measures analysis of variance wasconducted to test the treatment by time interactions, and the effect oftreatment over time on the behavior score. If an interaction oftreatment by time or overall treatment effect were significant at the0.05 level, the subgroup analysis would be conducted for the effect oftreatment at each time point at level 0.05. Otherwise, the subgroupanalysis would be considered as exploratory. The means (STD) and p-valuefor testing the difference between treated and control groups arepresented.

[0220] To evaluate the chemotactic activity of HUCB migration, counts ofintact cells were performed on the normal brain tissue extracts, andischemic brain tissue extracts at 6h, 24h and 1 week of ischemic onset.We tested the normality and equal variances of each outcome measure.Data transformation or permutation tests would be considered, if datawere ill behaved. The HUCB migration active were evaluated betweennormal tissue and ischemic tissues, respectively. The main effect wassignificant at level 0.05, then subgroup analysis would be consideredwith a significant effect at level of 0.05. The means (std) arereported.

[0221] Results

[0222] Functional tests: Rats treated with HUCB cells at 24 h afterstroke showed no treatment by time interaction for each treatment groupon each neurobehavioral score (p-value for interactions >0.13). Theoverall treatment effect was significant on NSS with p<0.01,adhesive-removal test p=0.04 and rotarod test p=0.01. FIGS. 3A, 3B and3C shows that treatment at one day after MCAo with HUCB significantlyimproved functional recovery at 14 days as evidenced by rotarod,adhesive-removal test and NSS scores (p<0.05). Rats treated with HUCB at7 days after stroke showed no treatment by time interaction onneurobehavioral scores with p-value for interaction at 0.88 for NSS,0.41 for the adhesive-removal test and 0.09 for the rotarod test scores.The overall treatment effect was significant only on NSS with p<0.05,and no treatment effect on the other tests (p=0.15 for adhesive removaltest and 0.55 for rotarod test score) was detected. FIGS. 4A, 4B, 4Cshow treatment at 7 days after MCAo with HUCB significantly improvedfunctional on NSS test (p<0.05) at 28 day and 35 day after MCAo comparedto control group. However, rotarod and adhesive-removal tests failed toshow a significant difference compared to control animals.

[0223] Histology: Within the 6 μm thick coronal sections stained withH&E, dark and red neurons were observed in the ischemic core of all ratssubjected to MCAo with and without donor transplantation at 14 and 35days after MCAo. No significant reduction of volume of ischemic damagewas detected in rats with donor treatment at 24 h and 7 days afterischemia, compared with control rats subjected to MCAo alone. Within thebrain tissue, identification of HUCB was characterized by MAB1281staining. HUCB survived and were distributed throughout the damagedbrain of recipient rats [FIG. 5]. MAB1281 reactive cells were observedin multiple areas of the ipsilateral hemisphere, including cortices andstriatum of the ipsilateral hemisphere. The vast majority of MAB1281reactive cells were located in the ischemic boundary zone [FIG. 5]. Fewcells were observed in the contralateral hemisphere. The data indicatethat HUCB cells delivered to brain via an intravenous route preferablymigrate into the injured tissue. Some MAB 1281 positive cells encirclevessels, and some cells were detected in the nuclei of the capillaryendothelial cells surrounding the injury area [FIG. 5].

[0224] Double staining immunohistochemistry of brain sections revealedthat some MAB 1281-positive cells were reactive for the astrocyte markerGFAP, neuronal markers NeuN and MAP-2, for endothelial cell markerFVIII. The percentage of MAB1281 labeled expressed GFAP, NeuN, MAP-2 andFVIII proteins was (˜6)%, (˜3)%, (˜2%) and (˜8%), respectively.

[0225] Ischemia Brain Tissue Extract Assay on HUCB Migration:

[0226] HUCB cells migrate in the presence of normal brain tissue andischemic tissue obtained at 6h, 24h and 1w after MCAo. A significantincrease in HUCB migration activity was detected in the presence ofischemic cerebral tissue harvested at 24 h after the onset of stroke(p<0.01). A trend of increase in HUCB migration activity was apparent ontissue harvested at 6 hour and 1 week after MCAo (p>0.09) compared toHUCB migration activity measured in the presence of on normalnon-ischemic brain tissue.

[0227] Results/Conclusions

[0228] The above-described experiments reveal that at 14 days and 35days after transplantation, intravenously injected HUCB were found inthe brain, and significantly more MAB 1281 positive cells were found inthe ipsilateral hemisphere than in the contralateral hemisphere. Manycells migrated into the boundary zone of ischemic brain and some cellssurrounded vessels. HUCB survive, and some express of cell-type-specificmarker GFAP, NeuN and MAP-2. Most important, a significant improvementin functional outcome on motor, sensory and modified NSS tests was foundin animals given HUCB intravenously at 1 day after stroke. In vitro, ourdata showed there was significant HUCB migration activity in thepresence of ischemic cerebral tissue harvested at 24 h after MCAo(p<0.01) compared to normal non-ischemic brain tissue. The HUCBtreatment at ischemia 24h promoted more HUCB migration into ischemicbrain that may facilitate to functional recovery after MCAo.

[0229] In this study, it is shown that intravenous infusion of HUCBenter brain, survive, differentiate and reduce neurological deficitsafter stroke. In the study, a small percentage of HUCB cells expressedproteins phenotypic of neuronal-like cells. Functional recovery wasfound within days after administration HUCB.

[0230] It was also shown that more HUCB were found in the lesionedhemisphere than in the intact Hemisphere as well as that ischemic braintissue extracts induced migration of HUCB, suggesting that ischemiainduced chemotactic factors facilitate UCB migration.

[0231] The results described herein show that HUCB treatment at 24 hafter MCAo in the present studies produced significant improvedfunctional recovery (motor rotarod, somatosensory adhesive-removal testand NSS scores) after stroke. Treatment with HUCB at 7 days after MCAoshowed functional recovery only on NSS test after MCAo. However, rotarodand somatosensory adhesive-removal test did not shown significantrecovery. The treatment benefit of HUCB, thus, may depend on the time oftreatment. The treatment benefit may be interrelated to the migrationactivity of HUCB. A significant increase in HUCB migration activity wasdetected in the presence of ischemic cerebral tissue harvested at 24 hafter MCAo. Treatment with HUCB at ischemic early may promote HUCBmigration into ischemic brain and facilitate functional recovery afterMCAo.

[0232] Almost 25% of cord blood harvests rapidly give rise to awell-established layer of fibroblastoid (MIC) cells. The rapid growth ofthese cells seems to be sustained by a population of (self-renewing)quiescent (G)) cells. MPC have large ex vivo expansion capacity as wellas on their differentiation potential cord blood-derived MPCs can bevisualized as attractive targets for cellular or gene transfertherapeutic options.

[0233] In conclusion, the experiments presented have shown thatintravenously administrated HUCB survive, migrate and improve functionalrecovery after stroke. Although the mechanism is unclear, the describedexperiments support the use of umbilical cord blood derived neural cellsfor the treatment of stroke.

Example Parenteral Administration of Human Umbilical Cord Blood inReducing Neurological Deficits After Traumatic Brain Injury

[0234] Materials and Methods

[0235] Preparation of Human Umbilical Cord Blood for Injection. Thehuman umbilical cord blood used was a gift from Cryocell International,INC. (Clearwater, Fla.). The specimen was stored in liquid nitrogen andthe cells were restored at 37° C. After centrifugation at 1000 rpm/minfor 10 min at 4° C., the supernatant was removed and the cells werewashed with 0.1 M PBS two times. 30 ul of the cell suspension was mixedwith 30 ul of 0.4% trypan blue stain and the number of the viable cellswas counted with a hemacytometer and a counter under a phase contrastmicroscope. The total number of the harvested cells was calculated andthe final dilution was 2×10⁶ cells in 300 μl saline.

[0236] Controlled Cortical Injury Animal Model and the Injection ofHUCB. Wistar rats were anesthetized with 350 mg/kg body weight chloralhydrate, intraperitoneally. Rectal temperature was controlled at 37° C.with a feedback regulated water-heating pad. A controlled corticalimpact device was used to induce the injury. Rats were placed in astereotactic frame. Two 10 unn diameter eraniotomies were performedadjacent to the central suture, midway between lamda and bregma. Thesecond craniotomy allowed for movement of cortical tissue laterally. Thedura was kept intact over the cortex. Injury was induced by impactingthe left cortex (ipsilateral cortex) with a pneumatic piston containinga 6 mm diameter tip at a rate of 4 m/s and 2.5 mm of compression.Velocity was measured with a linear velocity displacement transducer.

[0237] Twenty four rats subjected to TBI were divided into three groups.Experimental group (n=8): 24 hours after TBI, rats were slowly injectedover a 10 minute duration with 2×10⁶ cells in 300 gl saline via a tailvein. Placebo control group (n=8): 24 hours after TBI, rats were slowlyinjected over a 10 minute duration with 300 gl saline via a tail vein.TBI only group (n−−8): the rats only were subjected to TBI and notreatment. All rats were killed 28 days after the treatment.

[0238] Tissue Preparation. (1) Paraffin sections: Four animals from eachgroup were euthanized with an overdose of ketamine and xylazineadministered intraperitoneally and perfused with intra-cardiacheparinized saline followed by 10% buffered formalin. The brains,hearts, lungs, livers, kidneys, spleens, muscle and bone marrow wereremoved and stored in 10% buffered formalin for 24 hours. Seven standard2 mm thick blocks were cut on a rodent brain matrix and then embeddedwith paraffin. Two millimeter thick blocks of the other organs were alsocut and embedded with paraffin. A series of adjacent 6 gm thick sectionswere cut and a section of each block of the brain and other organs wasstained with H&E. Standard H&E staining was employed for morphologicalanalysis under light microscopy. (2) Vibratome sections: An additionalfour rats from each group received the intravenous administration of 1ml of saline containing fluorescein isothiocyanate (FITC)-dextran (50mlg/ml, 2×10⁶ molecular weight; Sigma, St. Louis, Mo.). This dyecirculated for 1 min, after which the anesthetized rats were killed bydecapitation. The brains were rapidly removed from severed heads andplaced in 4% paraformaldehyde at 4° C. for 48 hr. Coronal sections (100[tm) were cut on a vibratome.

[0239] Immunohistochemistry. Single staining was performed foridentification of HUCB cells using a primary mouse anti-human nucleimonoclonal antibody (MAB1281) and secondary Cy5-conjugated F (ab′)2Fragment rabbit anti-mouse IgG in the coronal sections of all organs.Double staining was also performed on coronal cerebral sections. Brainssections were initially stained for neuronal markers, NeuN and MAP-2, oran astrocytic marker, glial fibrillary acidic protein (GFAP), with thecorrespondence primary antibodies and the secondary FITC-conjugated F(ab′)2 fragment, and subsequently double stained with primary MAB1281antibody and second antibodies of Cy5-conjugated-F(ab′)2 fragment foridentification of human umbilical cord blood cells. Briefly, 6 m thicksections from TBI, TBI+saline and TBI+HUCB groups were deparaffinizedand the sections were put in boiling citrate buffer (pH=6) in amicrowave oven for 10 min for identification of neurons. After coolingat room temperature, the sections were incubated in 0.1% saponin-PBS at4° C. overnight for mAb NeuN (dilution 1: 400, Chemicon) and MAP-2(dilution 1: 400, Chemicon). Antimouse FITC-conjugated F (ab′)2 fragment(dilution 1: 20, Calbiochem, Calif.) was then added and incubated forone week. To identify astrocytes, the sections were treated with 0.1%pepsin 37° C. for 15 min and then pAb GFAP (dilution 1:400, Dakopatts)was added. The sections were incubated with antirabbit FITC-conjugated F(ab′)2 fragment (dilution 1: 20, Calbiochem, Calif.) for one week. Theabove sections stained with FITC-conjugated F (ab′) fragment weresubsequently processed for identification of a human cellular nucleiantigen with a primary mouse anti-human nuclei monoclonal antibody,MAB1281 (dilution, 1: 200) and a Cy5-conjugated F (ab′)2 fragment rabbitanti-mouse IgG (dilution, 1: 20). The slides were analyzed using afluorescent microscope (Olympus, BH-2). Negative control sections fromeach animal received identical staining preparation, except that theprimary antibodies or the secondary antibodies were omitted.

[0240] Three-dimensional image acquisition. In order to observe therelation of the donor's cells with the cerebral vessels, the vibratomesections were analyzed with a Bio-Rad (Cambridge, Mass.) MRC 1024 (argonand krypton) laser-scanning confocal imaging system mounted onto a Zeissmicroscope (Bio-Rad). With the FITC-perfused tissue samples from eachgroup, 10 vibratome sections from interaural 6.38 mm to interaural 1.0nun (Paxions and Watson, 1986) at 2 mm interval were screened at 488 umunder a 10× objective lens. Sections stained with the MAB antibody (Cy5)were excited by a laser beam at 647 nm.

[0241] Estimates of Cell Number. For measurement of MAB 1281 reactivecells, an average nmber of five equally spaced slides (approximately100p. m interval) were obtained from each brain block and MAB 1281reactive cells were counted within the seven 2 mm thick blocksencompassing the forebrain. Nine slides from each of these blocks werefirst stained with FITC staining for identification NeuN (3 slides),MAP-2 (3 slides) and GFAP (3 slides), and were followed by Cy5 stainingfor identification of HUCB cells. The number of the MAB 1281 reactivecells expressing NeuN, MAP-2 and GFAP were counted, respectively, usingfluorescent microscopy within all seven blocks. In order to reducebiases introduced by sampling parameters, all sections for MAB 1281identification from rats were stained simultaneously. The criteria forMAB 1281 positive cells were defined before the cells were counted byobservers blinded to the individual treatment. All MAB 1281 reactivecells were counted throughout the coronal sections.

[0242] Neurological Functional Evaluation. Neurological motormeasurement was performed using an accelerating Rotarod-motor test. Therats were placed on the accelerating Rotarod treadmill (Lab-lineinstruments, INC) and the rat's task was to walk and maintain itsequilibrium on the rotating rod that rotates at a gradually increasingspeed. When the rat falls off the rod, a plate trips and a liquidcrystal records the endurance time in seconds. All rats were pre-trainedwith five trials (warm up trials) performed daily for 3 days prior toTBI to ensure stable baselines. After TBI and TBI followingadministration of HUCB or saline, the rats were tested on days 1, 4, 7,14 and 28 until sacrifice. The motor test data are shown as a percentageof an average of five trials on the rotarod test compared with theinternal baseline values.

[0243] Twenty four hours after TBI or administration of HUCB or saline,all rats were evaluated using the neurological severity scores (NSS).NSS is a composite of the motor (muscle status, abnormal movement),sensory (visual, tactile and proprioceptive) and reflex tests. One pointwas given for failure to perform a task. Thus, the higher score, themore severe is injury, with a maximum of 14 points. Rats werereevaluated on days 1, 4, 7, 14 and 28 after the treatment. Allmeasurements were performed by observers blinded to individualtreatment.

[0244] Statistical Analysis. NSS and Rotarod tested scores were measuredbefore injury and at 1, 4, 7, 14 and 28 days after TBI. The numbers ofMAB 1281 reactive cells were counted at 28 days after treatment. We wereprimarily interested in the effect of HUCB on the recovery of NSS. Theanalysis began by testing the difference in means of NSS between the twocontrol groups. If there was no difference between the two controls at0.05 level, the two control groups were combined to increase the power.The analysis of covariance for ANOVA (repeated measures) was conductedto test the treatment by time interactions, and the effect of treatmentover time. If an interaction of time by time was detected at 0.10 level,then the subgroup analysis was conducted for the effect of treatment ateach time point at level 0.05. Otherwise, the subgroup analysis wasconsidered as exploratory. The same analysis approach was used toanalyze the outcome of Rotarod test score. Paired-t test was used totest the difference in means of cell counts between the injuredhemisphere and the control hemisphere.

[0245] Results

[0246] Histological analysis of organs. Sections from the blocks ofbrain and organs were stained with H&E staining for the generalhistopathologieal evaluation. The architectural integrity of all organsanalyzed under light microscopy was not disrupted except for the initialmechanical injury of the brain. Bleeding, invasion of white cells,inflammatory response and neoplasm were not observed on any slides asidefrom brain.

[0247] Distribution of MAB 1281 positive cells. No MAB 1281 positivecells were observed in the slides from only TBI and TBI+saline groupswhich did not receive the injection of HUCB. Large numbers of MAB 1281positive cells were found in the vessels of the brain, heart, lung,liver, kidney, spleen, muscle and even bone marrow of the rats receivingthe injection of HUCB. A few scattered MAB 1281 positive cells werefound in the parenchyma of these organs. In brain, MAB 1281 labeledcells were observed in the boundary zone of the injured area, cortex,striamm and corpus callosum of the ipsilateral hemisphere. The MAB 1281positive signals were detected in the nuclei of the capillaryendothelial cells surrounding the injured area. Using laser confocalmicroscopy, the implanted cells were confirmed to be integrated intosprouting vessels in the boundary zone of the injured area. The totalnumber of MAB 1281 positive cells migrating into the parenchyma of boththe ipsilateral and contralateral hemispheres of the brain was countedand analyzed in the TBI+HUCB group. The numbers of MAB 1281 positivecells in the ipsilateral hemisphere (43,597±4265) were significantlygreater than those in the contralateral hemisphere (13,742±6471, p<0.05). The data indicate that HUCB cells delivered to brain via anintravenous route preferably migrate into the injured tissue.

[0248] Phenotypical Identification of MAB 1281 reactive cells. Doublefluorescent staining showed that some MAB 1281 positive cells expressedneuronal markers, NeuN and MAP-2, and an astrocytic marker, GFAP. Thesedouble-labeled cells were observed only in the ipsilateral hemispheresof the rats in the HUCB treated group. Most of these positive cells werelocated in the boundary zone of the injured area. 6.9±1.3% of MAB 1281labeled cells in the ipsilateral hemispheres in the HUCB treated groupexpressed NeuN. 5.8±2.4% expressed MAP-2 and 9.7+−2.8% expressed GFAP.These data demonstrate that some implanted cells express neuronal andastrocytic phenotypes.

[0249] Neurological and Motor Function Evaluation. Two days after TBI,significantly lower scores of Rotarod test and significantly higherscores of NSS in three groups compared to preinjury were found. RotarodTest scores were significantly improved in TBI+HUCB group (138.0−+11.3%and 155.2−+16.2%) when compared with TBI (118.5±17.0% and 129.2±12.2%)and TBI+saline group (117.2+−13.6% and 133.2±10.7%. p<0.05) at days 14and 28 aRer administration of HUCB. The neurological severety scoreswere also significantly improved in TBI+HUCB group (4.2+−1.3 and 3±0.8)when compared with TBI group (7.5±1.73 and 6.3±1.3) and TBI+saline group(7.3±0.9 and 5.75±0.9), p<0.05) at days 14 and 28 after the injection.The results indicate that intravenous administration of HUCB 24 hoursafter TBI reduce the motor neurological functional deficits caused byTBI.

[0250] Conclusions

[0251] The major findings of the above-described experiments were: (1)HUCB cells injected intravenously enter brain by day 28 after HUCB celladministration; (2) intravenous injection ogHUCB reduces motor andneurological deficits by days 14 and 28 after administration; (3) thecells migrating into the parenchyma of the brain express the neuronalmarkers, NeuN and MAP-2, and the astrocytic marker, GFAP; (4) HUCB cellsintegrated into the vascular wall within target organ; (5) these cellsare also present in other organs and primarily localize to the vessels,without any obvious adverse effects. Our data suggest that intravenousadministration of HUCB may be useful in the treatment of TBI.

[0252] These data demonstrate that a few injected ceils migrate into theparenchyma of the brain, heart, lung, kidney, liver, spleen, muscle andbone marrow. Because our study was designed to measure the effect ofHUCB administered intravenously on traumatic brain injury, the numbersof HUCB cells (MAB 1281 positive cells) present in the cerebralparenchyma were counted and analyzed in the TBI+HUCB group.Significantly more MAB 1281 positive cells were found in the ipsilateralhemisphere than in the contralateral hemisphere. This indicates that theinjected cells preferably migrate into the injured hemisphere,especially to the boundary zone of the injured area after TBI and thatnearly all of the MAB 1281 positive cells expressing NeuN, MAP-2 andGFAP were located in the ipsilateral hemisphere, demonstrating that themicro-environment of the brain after injury may benefit the induction ofthe differentiation of HUCB stem cells into the neural cell phenotype.

[0253] Two tests (Rotarod test and NSS) were used to measure theneurological behavioral responses to experimental injury in rats. TheRotarod Test is a well-established procedure for testing limb motorcoordination and balance aspects of motor performance in rats. The NSSis similar to the Rotarod Test and is an economical, simple and rapidtest for assessing mild motor, sensory and reflex deficits after TBI.These two tests are generally used for the evaluation of the effects ofthe drugs on the behavioral responses after TBI and stroke in animals.Fourteen and twenty eight days after intravenous administration of HUCB,the neurological behavioral deficits were significantly reduced in therats subjected to TBI in the above-described experiments. These dataindicate that intravenous administration of HUCB can effectively improvethe neurological outcome in rats after TBI and that intravenousadministration of HUCB to patients suffering damage to the brain and/orspinal cord represents a viable therapeutic approach for treating suchinjuries, including traumatic brain injury.

[0254] While the invention has been described hereinabove, care shouldbe taken not to limit the invention in a manner which is unintended andis inconsistent with the invention as set forth in the following claims.

1. Neural cells obtained by exposing pluripotent stem or progenitorcells obtained from umbilical cord blood to an amount of adifferentiation agent effective for changing the phenotype of said stemor progenitor cells to a neural phenotype.
 2. The cells of claim 1wherein said differentiation agent is selected from the group consistingof retinoic acid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF,TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT-SCF, interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signalling molecules and mixtures, thereof.
 3. The cellsof claim 1 wherein said differentiation agent is a mixture of retinoicacid and NGF.
 4. A method of producing neural cells from umbilical cordblood comprising: a. obtaining a sample of mononuclear cells from saidumbilical cord blood; and b. growing said mononuclear cells from step ain a culture medium containing an effective amount of a differentiationagent for a period sufficient to change the phenotype of said stem orprogenitor cells to neural.
 5. The method according to claim 4 whereinsaid differentiation agent is selected from the group consisting ofretinoic acid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF,TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon,triiodothyronine, thyroxine, erthyopoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signalling molecules and mixtures, thereof.
 6. Themethod according to claim 4 wherein said differentiation agent is amixture of retinoic acid and NGF.
 7. The method according to claim 5wherein said neuronal cells are selected from the group consisting ofmesencephalic cells and striatal cells.
 8. A method of producing neuralcells from umbilical cord blood comprising: a. obtaining a sample ofmononuclear cells from said umbilical cord blood; b. selecting for andisolating a sample of pluripotent stem or progenitor cells within saidsample; and c. growing said stem or progenitor cells from step b in aculture medium containing an effective amount of a differentiation agentfor a period sufficient to change the phenotype of said stem orprogenitor cells to neural.
 9. The method according to claim 8 whereinsaid selecting and isolating step b is carried out using a magnetic cellseparator to separate out cells containing a CD marker.
 10. The methodaccording to claim 8 wherein said differentiation agent is selected fromthe group consisting of retinoic acid, fetal or mature neuronal cells,BDNF, GDNF, NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT,interferon, triiodothyronine, thyroxine, erthyopoietin, thrombopoietin,silencers, SHC, neuroproteins, proteoglycans, glycoproteins, neuraladhesion molecules, cell signalling molecules and mixtures, thereof. 11.A method of producing neural cells from umbilical cord blood comprising:a. obtaining a sample of mononuclear cells from said umbilical cordblood; b. growing said mononuclear cells from step b in a culture mediumcontaining an effective amount of a differentiation agent for a periodsufficient to change the phenotype of pluripotent stem or progenitorcells within said mononuclear cells to neural; and c. selecting for andisolating said neural cells from said sample of pluripotent stem orprogenitor cells within said sample by essentially eliminating from saidsample mononuclear cells having a CD marker.
 12. The method according toclaim 11 wherein said selecting and isolating step c is carried outusing a magnetic cell separator to separate out cells containing a CDmarker.
 13. The method according to claim 11 wherein saiddifferentiation agent is selected from the group consisting of retinoicacid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF, TGF, CNTF,BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon, triiodothyronine,thyroxine, erthyopoietin, thrombopoietin, silencers, SHC, neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, cell signallingmolecules and mixtures, thereof.
 14. The method according to claim 13wherein said neuronal cells are selected from the group consisting ofmesencephalic cells and striatal cells.
 15. A method of producing asample of enriched neural cells from a sample of mononuclear cellsobtained from umbilical cord blood comprising: a. subjecting themononuclear cells to an amount of an anti-proliferating cell agenteffective to eliminate essentially all proliferating cells from saidmononuclear cell sample; b. exposing the remaining non-proliferatingcells from step a to a mitogen to provide a population of differentiatedcells and quiescent cells comprising a population of pluripotent stem orprogenitor cells; c. growing said population of said differentiatedcells and quiescent cells from step b to selectively grow said quiescentcells to the essential exclusion of differentiated cells.
 16. The methodaccording to claim 15 comprising the further step of incubating a cellpopulation obtained from step c to a differentiation agent effective toinduce a neural phenotype in said pluripotent stem or progenitor cells.17. The method according to claim 11 wherein said anti-proliferativecell agent is Ara-C.
 18. The method according to claim 11 wherein saidmitogen is selected from the group consisting of epidermal growth factorand pokeweed mitogen.
 19. The method according to claim 12 whereindifferentiation agent is selected from the group consisting of retinoicacid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF, TGF, CNTF,BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon, triiodothyronine,thyroxine, erthyopoietin, thrombopoietin, silencers, SHC, neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, cell signallingmolecules and mixtures, thereof.
 20. The method according to claim 15wherein said retinoic acid is selected from 9-cis retinoic acid, alltransretinoic acid and mixtures, thereof.
 21. The method according toclaim 3 wherein said neural cells are used in allogeneictransplantation.
 22. The method according to claim 5 wherein said neuralcells are used in allogeneic transplantation.
 23. The method accordingto claim 7 wherein said neural cells are used in allogeneictransplantation.
 24. The method according to claim 9 wherein said neuralcells are used in allogeneic transplantation.
 25. The method accordingto claim 11 wherein said neural cells are used in allogeneictransplantation.
 26. The method according to claim 15 wherein saidneural cells are used in allogeneic transplantation.
 27. A method oftreating a damaged brain or spinal cord comprising transplanting intosaid brain or spinal cord an effective of number neural cells accordingto claim
 1. 28. A method of treating a patient with a neurodegenerativedisease comprising administering an effective number of neural cellsaccording to claim 1 to said patient.
 29. The method according to claim24 wherein said neurodegenerative disease is selected from the groupconsisting of Parkinson's disease, Alzheimer's disease, multiplesclerosis (MS), Tay Sach's disease, Rett Syndrome, lysosomal storagedisease, ischemia, spinal cord damage, ataxia, alcoholism, amyotrophiclateral sclerosis, schizophrenia and autism.
 30. A method of treating apatient with a neurodegenerative disease comprising transplanting aneffective number of neural cells obtained according to the method ofclaim 3 to said patient.
 31. The method according to claim 26 whereinsaid neurodegenerative disease is selected from the group consisting ofParkinson's disease, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 32. A method of treating a patientwith a neurodegenerative disease comprising administering an effectivenumber of neural cells obtained according to the method of claim 5 tosaid patient.
 33. The method according to claim 28 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 34. A method of treating a patientwith a neurodegenerative disease comprising administering an effectivenumber of neural cells obtained according to the method of claim 7 intosaid patient.
 35. The method according to claim 30 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 36. A method of treating a patientwith a neurodegenerative disease comprising administering an effectivenumber of neural cells obtained according to the method of claim 9 intosaid patient.
 37. The method according to claim 32 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 38. The method according to claim33 wherein said ischemia is caused by a stroke or heart attack in saidpatient.
 39. A method of treating a patient with a neurodegenerativedisease comprising administering an effective number of neural cells inumbilical cord blood or a mononuclear cell fraction thereof to saidpatient.
 40. The method according to claim 39 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 41. A method of treating a patientwith a neurodegenerative disease other than amyotrophic lateralsclerosis comprising administering an effective number of neural cellsto said patient.
 42. The method according to claim 41 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease (beta hexosaminidase deficiency), Rett Syndrome, lysosomalstorage disease ischemia, spinal cord damage, traumatic brain injury,ataxia, alcoholism, schizophrenia and autism.
 43. A compositioncomprising umbilical cord blood or a mononuclear cell fraction, thereof,in combination with an effective amount of at least one neuraldifferentiation agent.
 44. The composition according to claim 40 furthercomprising a cell medium to which said differentiation agent is added.45. The composition according to claim 40 wherein said differentiationagent is selected from the group consisting of retinoic acid, fetal ormature mesencephalic or striatal cells brain derived neurotrophic factor(BDNF), glial derived neurotrophic factor (GDNF), glial growth factor(GFF), nerve growth factor (NGF), fibroblast growth factor (FGF),transforming growth factors (TGF), ciliary neurotrophic factor (CNTF),bone-morphogenetic proteins (BMP), leukemia inhbitory factor (LIF),glial growth factor (GGF), tumor necrosis factors (TNF), interferon,insulin-like growth factors (IGF), colony stimulating factors (CSF), KITreceptor stem cell factor (KIT-SCF), interferon, triiodothyronine,thyroxine, erythropoietin, thrombopoietin, glial-cell missing silencerfactor, neuron restrictive silencer factor,SRC-homology-2-domain-containing transforming protein, neuroproteins,proteoglycans, glycoproteins and neural adhesion molecules.
 46. Thecomposition according to claim 40 wherein said differentiation agent isselected from the group consisting of retinoic acid, fetal or maturemesencephalic or striatal cells, brain derived neurotrophic factor(BDNF), glial derived neurotrophic factor (GDNF), glial growth factor(GFF), nerve growth factor (NGF) and mixtures, thereof.
 47. Thecomposition according to claim 40 wherein said differentiation agent isselected from the group consisting of mixtures of retinoic acid, brainderived neurotrophic factor (BDNF), glial derived neurotrophic factor(GDNF), glial growth factor (GFF) and nerve growth factor (NGF).
 48. Thecomposition according to claim 44 further comprising a cell medium towhich said differentiation agent is added.
 49. The composition accordingto claim 40 wherein said differentiation agent is a mixture of retinoicacid and nerve growth factor.
 50. A method of producing a pharmaceuticalcomposition comprising a sample of mononuclear cells being enriched withcells having a neural phenotype marker, said method comprising: a.obtaining a sample of mononuclear cells from said umbilical cord blood;and b. growing said mononuclear cells from step a in a culture mediumcontaining an effective amount of a differentiation agent for a periodsufficient to change the phenotype of said stem or progenitor cells toneural; and c. combining said cells obtained from step b with apharmaceutically acceptable carrier, additive or excipient.
 51. Themethod according to claim 50 wherein said differentiation agent isselected from the group consisting of retinoic acid, fetal or matureneuronal cells, BDNF, GDNF, NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF,IGF, CSF, KIT, interferon, triiodothyronine, thyroxine, erthyopoietin,thrombopoietin, silencers, SHC, neuroproteins, proteoglycans,glycoproteins, neural adhesion molecules, cell signalling molecules andmixtures, thereof.
 52. The method according to claim 50 wherein saiddifferentiation agent is a mixture of retinoic acid and NGF.
 53. Themethod according to claim 50 wherein said neuronal cells are selectedfrom the group consisting of mesencephalic cells and striatal cells. 54.A method of producing a pharmaceutical composition comprising neuralcells obtained from umbilical cord blood comprising: a. obtaining asample of mononuclear cells from said umbilical cord blood; b. selectingfor and isolating a sample of pluripotent stem or progenitor cellswithin said sample; c. growing said stem or progenitor cells from step bin a culture medium containing an effective amount of a differentiationagent for a period sufficient to change the phenotype of said stem orprogenitor cells to neural.; and d. combining said cells obtained fromstep b with a pharmaceutically acceptable carrier, additive orexcipient.
 55. The method according to claim 54 wherein said selectingand isolating step b is carried out using a magnetic cell separator toseparate out cells containing a CD marker.
 56. The method according toclaim 54 wherein said differentiation agent is selected from the groupconsisting of retinoic acid, fetal or mature neuronal cells, BDNF, GDNF,NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon,triiodothyronine, thyroxine, erthyopoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signalling molecules and mixtures, thereof.
 57. A methodof producing a pharmaceutical composition comprising neural cellsobtained from umbilical cord blood comprising: a. obtaining a sample ofmononuclear cells from said umbilical cord blood; b. growing saidmononuclear cells from step b in a culture medium containing aneffective amount of a differentiation agent for a period sufficient tochange the phenotype of pluripotent stem or progenitor cells within saidmononuclear cells to neural; and c. selecting for and isolating saidneural cells from said sample of pluripotent stem or progenitor cellswithin said sample by essentially eliminating from said samplemononuclear cells having a CD marker; and d. combining said neural cellsisolated from step c with a pharmaceutically acceptable carrier,additive or excipient.
 58. The method according to claim 57 wherein saidselecting and isolating step c is carried out using a magnetic cellseparator to separate out cells containing a CD marker.
 59. The methodaccording to claim 57 wherein said differentiation agent is selectedfrom the group consisting of retinoic acid, fetal or mature neuronalcells, BDNF, GDNF, NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF,KIT, interferon, triiodothyronine, thyroxine, erythropoietin,thrombopoietin, silencers, SHC, neuroproteins, proteoglycans,glycoproteins, neural adhesion molecules, cell signalling molecules andmixtures, thereof.
 60. The method according to claim 57 wherein saiddifferentiation agent is a mixture of retinoic acid and nerve growthfactor.
 61. The method according to claim 57 wherein said neuronal cellsare selected from the group consisting of mesencephalic cells andstriatal cells.
 62. A method of treating a patient for aneurodegenerative disease selected from the group consisting of multiplesclerosis (MS), Tay Sach's disease (beta hexosaminidase deficiency),Rett Syndrome, and lysosomal storage disease said method comprisingadministering to said patient an effective amount of human umbilicalcord blood or a mononuclear cell fraction thereof to said patient. 63.The method according to claim 62 wherein said human umbilical cord bloodor said mononuclear cell fraction thereof is administered via aparenteral route of administration.
 64. A method of treating a patientin need thereof for a neurodegenerative disease other than amyotrophiclateral sclerosis, said method comprising administering an effectiveamount of human umbilical cord blood or a mononuclear cell fractionthereof to said patient.
 65. The method according to claim 64 whereinsaid neurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease, multiplesclerosis (MS), Tay Sach's disease, Rett Syndrome, lysosomal storagedisease, ischemia, spinal cord damage, traumatic brain injury, ataxia,alcoholism, schizophrenia and autism.
 66. A method of treating a patientin need thereof for a neurodegenerative disease comprising administeringan effective amount of neural cells to said patient in the absence of aradiation step or chemotherapeutic step which is used to impair bonemarrow production of hematopoietic cells.
 67. The method according toclaim 66 wherein neural cells are administered to said patient via aroute of administration selected from the group consisting ofintrathecal, intraventricular, intraparenchymal, intracisternal,intracranial, intrastriatal, and intranigral.
 68. The method accordingto claim 67 wherein said neurodegenerative disorder is selected from thegroup consisting of Parkinson's disease, Huntington's disease,Alzheimer's disease, multiple sclerosis, Tay Sach's disease, RettSyndrome, lysosomal storage disease, spinal cord damage, traumatic braininjury, ataxia, schizophrenia and autism.
 69. A method of treatingamyotrophic lateral sclerosis in a patient in need thereof, said methodcomprising administering an effective amount of human umbilical cordblood or a mononuclear cell fraction thereof to said patient in theabsence of a radiation step or chemotherapeutic step which is used toimpair bone marrow production of hematopoietic cells.